Ship inhibition to induce expression of granulocyte colony stimulating factor in a subject

ABSTRACT

The present invention relates to the use SHIP inhibitors to induce expression of granulocyte colony stimulating factor (G-CSF) in a subject, thereby promoting expansion of hematopoietic and mesenchymal stem cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional Patent Application Ser. No. 62/013,511, filed Jun. 17, 2014, the disclosure of which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to the use of SHIP inhibitors to induce expression of granulocyte colony stimulating factor (G-CSF) in a subject.

BACKGROUND OF THE INVENTION

Adult stem cells are capable of replenishing blood and immune cells, and rejuvenating tissues necessary to sustain life. The majority of adult stem cell populations can be found in the quiescent state and only proliferate in response to growth and stress signals. Manipulation of growth factor receptor pathways that sustain and promote cycling of stem cells has potential in clinical setting where stem cell numbers and function are compromised (e.g, chemotherapy and radiation treatments). One can envision such strategies facilitating improved outcomes for patients with various ailments ranging from bone marrow failure syndromes to cardiovascular disease.

Inositol phospholipid signaling plays a prominent role in hematopoietic stem cell (HSC) biology where Phosphatidylinositide 3-Kinase (PI3K), Phosphatase and tensin homolog (PTEN) and the SH2 domain containing inositol-5-phosphatase (SHIP1) regulate or promote quiescence, maintenance, and/or expansion of the HSC compartment.¹⁻⁴ Previously it was found that a significant expansion of HSC in the BM of SHIP1^(−/−) mice based on phenotypes enriched for long-term HSC (LT-HSC).⁵ SHIP1^(−/−) mice also exhibit increased production of cytokines or soluble niche factors that influence HSC homeostasis, such as, granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO) and matrix metallopeptidase 9 (MMP-9).⁶ Despite this phenotypic expansion, HSC from germline SHIP1^(−/−) mice exhibit defective repopulating activity in both competitive repopulating unit (CRU) and direct competition assays (DCA) that suggested SHIP1 signaling might have a cell intrinsic role in HSC function.^(5,7)

Analysis of HSC rendered SHIP1-deficient while resident in a SHIP1 competent niche failed to show a defect in either long-term, multi-lineage repopulating or self-renewal capacity, demonstrating that SHIP1 is not an intrinsic cell signaling component required for maintenance of HSC function.⁶ In addition, this study revealed that SHIP1 is expressed and has a functional role in the stromal cells and osteoblast compartments that support HSC.⁶ Subsequent analysis of mice with selective deletion of SHIP1 in mesenchymal stem cell (MSC) showed that SHIP1 plays a cell autonomous role in limiting MSC self-renewal by opposing the PI3K/Akt/β-catenin/Id2 axis that can drive MSC expansion.^(8, 9) A significant caveat of the above studies is that analysis of HSC and their niche components were performed in germline SHIP1^(−/−) mice, which suffer from significant mucosal inflammation that often contributes to the demise of mutant mice.¹⁰⁻¹² Whether induced or systemic, ablation of SHIP1 expression in adult mice leads to compromised HSC function, pneumonia, ileitis and death.^(6, 12)

There is a need to develop new methods of improving declining stem cell function in both therapy induced and genetically derived bone marrow failure syndromes.

The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a method of inducing expression of granulocyte colony stimulating factor (G-CSF) in a subject. This method involves administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor to the subject.

Promoting the expansion of adult stem cell populations offers the potenial to ameliorate radiation or chemotherapy-induced bone marrow failure and allows for expedited recovery for patients undergoing these therapies. Previous genetic studies suggested a pivotal role for SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) in limiting the size of the hematopoietic stem cell (HSC) compartment. In accordance with one aspect, the present invention involved determining small molecule SHIP1 inhibitors for use in the pharmacological expansion of the HSC compartment in vivo. As provided in more detail in the Examples herein, the present disclosure provides that treatment of mice with aminosteroid inhibitors of SHIP1 (SHIPi) more than doubles the size of the adult mesenchymal stem cell (MSC) compartment while simultaneously expanding the HSC pool six-fold. Consistent with its ability to target SHIP1 function in vivo, SHIPi also significantly increases plasma granulocyte colony stimulating factor (G-CSF) levels, a growth factor that supports proliferation of HSC. The present disclosure further shows that SHIPi induced G-CSF production mediates HSC and MSC expansion, as in vivo neutralization of G-CSF abrogates the SHIPi-induced expansion of both the HSC and MSC compartments. Due to its expansionary effect on adult stem cell compartments, SHIPi represents a novel strategy to improve declining stem cell function in both therapy induced and genetically derived bone marrow failure syndromes.

These and other objects, features, and advantages of this invention will become apparent from the following detailed description of the various aspects of the invention taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For purpose of illustrating aspects of the present invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. Further, as provided, like reference numerals contained in the drawings are meant to identify similar or identical elements.

FIGS. 1A-1I: SHIPi expands the HSC compartment. Flow cytometric quantitation of hematopoietic stem/progenitor populations in the BM of SHIPi and vehicle control mice. FIG. 1A: Total BM cellularity per femur/tibia pair as determined by hemocytometer. (n=5/group). FIG. 1B: Representative FACS analysis of Lin⁺Scal⁺Kit⁺ (LSK) in the BM of SHIPi and vehicle-treated control mice. (n=5/group). FIG. 1D: Representative FACS analysis of LT-HSC (LSKFlk2⁻CD34⁻), ST-HSC (LSKFlk2⁻CD34⁺), and MPP (LSKFlk2⁺CD34⁺) populations in WBM in SHIPi and vehicle-treated control mice. (n=5/group). Absolute numbers of LT-HSC (FIG. 1E), ST-HSC (FIG. 1F), and MPP populations in SHIPi and vehicle-treated control mice. (n=5/group) (FIG. 1G). FIG. 1H: Representative FACS analysis showing cell cycle state of BM LT-HSC based on Hoechst and Pyronin Y staining in SHIPi and vehicle-treated control mice. (n=5/group). FIG. 1I: Percentage distribution of LT-HSC (LSKCD34−) in G0, G1 and S+G2/M stages of the cell cycle. (n=5/group). The above analyses are representative of two independent comparisons of SHIPi vs. vehicle treated mice.

FIGS. 2A-2F: The SHIPi expanded HSC compartment retains normal long-term lineage repopulation and self-renewal potential. CRU assays were established where equal numbers of BM cells from SHIPi and vehicle-treated donors were competed against each other in lethally irradiated CD45.1⁻CD45.2⁺ heterozygous hosts. FIG. 2: Global repopulation of peripheral blood mononuclear cells (PBMC) in primary CRU hosts by SHIPi and vehicle-treated hematopoietic stem/progenitors (4 months post-transplant) (n=5/group). FIG. 2B: Relative PBMC reconstitution of representative hematolymphoid lineages (T lymphoid, B lymphoid, NK cells and myeloid cells) by SHIPi and vehicle treated hematopoietic stem/progenitors (4 months post-transplant). (n=5/group). FIG. 2C: Frequency of CRU in the BM of SHIPi and vehicle control donors. (n=5/group). FIG. 2D: Absolute CRU activity (per hind limb pair) in SHIPi and vehicle control mice after accounting for total BM cellularity. WBM cells were harvested from primary CRU donors and serial transferred to lethally irradiated CD45.1+CD45.2+ heterozygous hosts to create secondary CRU mice. (n=5/group). FIG. 2E: Global repopulation of PBMC in secondary CRU hosts by SHIPi and vehicle-treated hematopoietic stem/progenitors (1 month post-transplant) (n=5/group). FIG. 2F: Relative PBMC reconstitution of representative hematolymphoid lineages (T lymphoid, B lymphoid, NK cells, and myeloid cells) by SHIPi and vehicle-treated hematopoietic stem/progenitors in secondary CRU hosts (1 month post-transplant). (n=5/group). The above results are representative of two independent primary and secondary CRU assays for SHIPi vs. vehicle-treated BM donors.

FIGS. 3A-3B: SHIPi increases the frequency of mesenchymal stem cells present in the BM compartment. WBM cells were prepared by crushing a femur/tibia pair as described in the Methods. The cells were then stained and analyzed by flow cytometry for surface expression of PDGFRα, CD51, a hematolymphoid lineage marker panel (Lin) and CD31. FIG. 3A: Representative contour plots for SHIPi and vehicle control mice used to identify pluripotent PDGFRα⁺CD51⁺ MSC as defined by Brooks et al.¹³ The contour plots represent all viable CD31⁻CD45⁻Lin⁻BM cells. FIG. 3B: Box-and-whisker plots showing the frequency of PDGFRα⁺CD51⁺CD31⁻CD45⁻Lin⁻ MSC in SHIPi and vehicle control mice. (n=5/group). The above analyses are representative of two independent comparisons of SHIPi vs. vehicle treated mice.

FIGS. 4A-4D: Neutralization of G-CSF prevents the SHIPi-mediated increase in BM cellularity and expansion of the HSC compartment. FIG. 4: SHIPi increases G-CSF production in vivo. ELISA quantization of G-CSF in the indicated groups of mice. (n=5/group). FIG. 4B: Rescue of BM cellularity by anti-G-CSF as determined by hemocytometer counts. (n=5/group). FIG. 4C: Absolute LT-HSC numbers restored to normal frequency after administration of anti-G-CSF (n=5/group). FIG. 4D: Anti-G-CSF treatment abrogates the significant increase in MSC frequency observed in SHIPi treated mice as compared to vehicle controls. (n=5/group). The above analyses are representative of two independent comparisons of SHIPi vs. vehicle treated mice.

FIGS. 5A-5B: Rapid recovery of hematopoiesis in radiation induced bone marrow failure model. Post sub-lethal irradiation (550 Rads), mice were treated with SHIPi or vehicle for one week as described in Brooks et al.¹³ and then lymphocyte (n=5/group) (FIG. 5A) and WBC lymphocyte (n=5/group) (FIG. 5B) numbers were monitored via Hemavet (950FS DREW) for several weeks. The above analyses are representative of two independent comparisons of SHIPi vs. vehicle treated mice.

FIGS. 6A-6F: Characterizations of the soluble SHIPi compound K118. FIG. 6A: Fluorescence Polarization (FP) assay comparing SHIP1 enzymatic inhibitory activity between 3AC and K118 (IC50-13.87). In the remaining panels we show for K118 and H₂O treated mice (as indicated). FIG. 6B: Representative box and whisker graph depicting serum levels of G-CSF (n=4/group); FIG. 6C: Flow cytometric analysis comparing LT-HSC (LSKFlk2−CD34−), ST-HSC (LSKFlk2−CD34+), and MPP (LSKFlk2⁺CD34⁺); FIG. 6D: Absolute increase in BM LT-HSC, ST-HSC, and MPP (n=5/group). Primary CRU assay illustrating global and lineage reconstitution one month post transplant; Absolute increase in repopulating units (RU). FIG. 6E: Representative contour plots for PDGFRa⁺CD51⁺ MSC (after excluding CD45⁺, CD31⁺, Lin⁺ cells); FIG. 6F: Box and whiskers graph illustrating the percentage of PDGFRa⁺CD51⁺ MSC (n=5/group); Representative images of CFU-F assays used to measure CFU-F frequencies in BM cells of treated mice and Box and whiskers graphs indicating CFU-F frequency in treated mice. All of the above comparisons are representative of two independent experiments.

FIGS. 7A-7F: SHIPi significantly increases primitive and differentiated blood cell frequency without compromising homing. Mice were treated daily for 5 days and then on the 6^(th) day the WBM frequency of LSK (FIG. 7A), LSKF⁻34⁻ (LT-HSC) (FIG. 7B), LSKF⁻CD34⁺ (ST-HSC) (FIG. 7C) and LSKF⁺CD34⁺ (MPP) (FIG. 7D) in SHIPi treated vs. vehicle controls. FIGS. 7E-7F: Medium fluorescent intensity (MFI) of CXCR4 on Lin⁻Scal⁺c-Kit⁺Flk2⁻ HSC in the BM of SHIPi and vehicle treated mice (n=5/group). The above analyses are representative of two independent comparisons of SHIPi vs. vehicle treated mice.

FIGS. 8A-8D: SHIPi improves the initial and long-term reconstituting capacity of hematopoietic/stem progenitor grafts. FIG. 8A: Results of FACS analysis on PBMC from the primary CRU assay hosts as described in FIG. 6. Global and lineage reconstitution of CD3e⁺ T cells (FIG. 8B), CD19⁺ B cells (FIG. 8C) and Macl⁺/Grl⁺ myeloid cells (FIG. 8D) at the indicated times post-transplant for SHIPi- and vehicle-derived BM stem/progenitors. (n=5/group). The results are representative of two independent primary CRU assays for SHIPi- vs. vehicle-treated donors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to new uses of SHIP inhibitors for therapeutic purposes. More particularly, the present invention relates to the use of SHIP inhibitors, including, without limitation, SHIP1 and/or pan-SHIP1/2 inhibitors, for the inhibition of SHIP to induce expression of granulocyte colony stimulating factor (G-CSF) to treat various diseases or conditions.

In one aspect, the present invention relates to a method of inducing expression of G-CSF in a subject, where the method involves administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor to the subject.

Suitable SHIP inhibitors are described in more detail herein below. Further, suitable SHIP inhibitors and synthetic pathways for various-suitable SHIP inhibitors are described in WO 2011/127465 A2 to Kerr et al., WO 2015/003003 A1 to Kerr et al., the disclosures of which are incorporated by reference herein in their entirety.

According to various embodiments, the substance suitable for the instant invention can be a nucleic acid, such as a genetic construct or other genetic means directing expression of an antagonist of SHIP function. Nucleic acid molecules suitable for the inventive method include anti-sense polynucleotides, other polynucleotides that bind to SHIP mRNA, recombinant retroviral vector, or a combination thereof. A preferred genetic construct of the invention comprises a gene delivery vehicle, a recombinant retroviral vector, or a combination thereof. In a preferred embodiment, the substance that inhibits SHIP function is a nucleic acid that hybridizes to a SHIP mRNA.

In other embodiments, the substances suitable for the instant invention may also include peptidomimetic inhibitors of SHIP function, ribozymes, and an RNA aptamer, or a combination thereof.

Pharmaceutical agents or genetic therapies that reduce or eliminate SHIP activity and function encompass, but are not limited to the following: 1) small molecule inhibitors (preferably having a molecular weight of less than 10,000) of SHIP enzymatic activity (i.e. suicide substrates; competitive or non-competitive inhibitors of SHIP activity; RNA aptamers; or PIP 3, 4, or 5 analogs), 2) anti-sense oligonucleotides, 3) peptidomimetics, 4) ribozymes, 5) means for interfering with transcription and/or translation of SHIP RNA, or 6) genetic therapy comprising transfection with a dominant negative SHIP mutant. These agents and/or genetic therapies can exert their effects by preventing the recruitment of SHIP to complexes with other signal transduction components or to the plasma membrane where SHIP can access its inositol phospholipid substrates.

Within the present disclosure, the following terms are to be understood as follows:

An “isolated polypeptide” or “isolated polynucleotide” as used herein refers to a polypeptide or polynucleotide, respectively, produced in vivo or in vitro in an environment manipulated by humans using state of the art techniques of molecular biology, biochemistry and gene therapy. For example, an isolated polypeptide can be produced in a cell free system by automated peptide or polypeptide synthesis, in heterologous host cells transformed with the nucleic acid sequence encoding the polypeptide and regulatory sequences for expression in the host cells, and in an animal into which the coding sequence of the polypeptide has been introduced for expression in the animal. A polypeptide or polynucleotide is “isolated” for purposes herein to the extent that it is not present in its natural state inside a cell as a product of nature. For example, such isolated polypeptides or polynucleotides can be 10% pure; 20% pure, or a higher degree of purity.

The term “inositol polyphosphate 5-phosphatase” as used herein refers to a family of phosphatases each of which removes the 5 phosphate from inositol- and phosphatidylinositol-polyphosphates. The family of proteins is determined by the substrate specificity of these enzymes and by amino acid sequence homology. A description of some of the aspects of the family is provided in Jefferson and Majerus, J Biol Chem 270: 9370-77 (1995). The term “activated T cell” and “activated B cell” refers to T and B cells that have been stimulated, for example, with cytokines or growth factors, or which have had their antigen receptors cross-linked using antibodies, all of which events stimulate gene expression, cell proliferation or other responses in T and B cells.

The term “tyrosine phosphorylated” as used herein refers to the addition of a phosphate group at a tyrosine residue. Generally, tyrosine phosphorylation of polypeptides is associated with activation or inactivation of signaling pathways. Tyrosine phosphorylation is also associated with activation or inhibition of signaling molecules. Tyrosine phosphorylation of a polypeptide of the invention can occur in response to, for example, B or T cell activation. In some cases, binding to other polypeptides occurs before, after, or during they tyrosine phosphorylation of a polypeptide.

The term “apparent molecular weight” as used herein refers to the molecular weight of the protein or polypeptide as it migrates on a polyacrylamide gel under reducing or non-reducing conditions. The “apparent” molecular weight may be accounted for by glycosylations or other moieties that alter the molecular weight of the polypeptide alone.

The term “SHIP” as used herein refers to SH2-containing inositol-5-phosphatase. SHIP may have an apparent molecular weight of about 145 kDa and is expressed in at least hemopoietic cells. It contains an amino-terminal src-homology domain (SH2), a central 5′-phosphoinositol phosphatase domain, two phosphotyrosine binding consensus sequences, and a proline-rich region at the carboxyl tail.

The term a “means for inhibiting SHIP function” comprises genetic and non-genetic means for inhibiting SHIP function, and includes substances that inhibit SHIP functions.

Among the genetic construct inhibiting SHIP function are various “gene delivery vehicles” known to those of skill in the art, that facilitate delivery to a cell of, for example, a coding sequence for expression of a polypeptide, such as a SHIP inhibitor, an anti-sense oligonucleotide, an RNA aptamer capable of inhibiting SHIP enzymatic activity, an RNA aptamer capable of inhibiting a ribozyme, or another genetic construct of inhibiting SHIP activity known to those of skill in the art.

Among the non-genetic means inhibiting SHIP function are pharmaceutical agent, pharmaceutically acceptable salts thereof that are preferably administered in a pharmaceutically acceptable carrier.

According to preferred embodiments, substances suitable for the instant invention can be a nucleic acid, such as a genetic construct or other genetic means directing expression of an antagonist of SHIP function. Nucleic acid molecules suitable for the inventive method include anti-sense polynucleotides, other polynucleotides that bind to SHIP mRNA, recombinant retroviral vector, or a combination thereof. A preferred genetic construct of the invention comprises a gene delivery vehicle, a recombinant retroviral vector, or a combination thereof. In a preferred embodiment, the substance that inhibits SHIP function is a nucleic acid that hybridizes to a SHIP mRNA.

Preferred substances may also include peptidomimetic inhibitors of SHIP function, ribozymes, and an RNA aptamer, or a combination thereof.

Suitable substances for the instant invention may also be a low molecular weight substance having a molecular weight of less than about 10,000 that inhibits SHIP activity.

The cell to which said component or substance is delivered can be within a mammal, as in in vivo gene therapy, or can be removed from a mammal for transfection, or administration of a pharmaceutical agent, and can be subsequently returned to the mammal, as, for example, in ex vivo therapy or ex vivo gene therapy. The delivery vehicle can be any component or vehicle capable of accomplishing the delivery of a gene or substance to a cell, for example, a liposome, a particle, naked DNA, or a vector. A gene delivery vehicle is a recombinant vehicle, such as a recombinant viral vector, a nucleic acid vector (such as plasmid), a naked nucleic acid molecule such as a gene, a nucleic acid molecule complexed to a polycationic molecule capable of neutralizing the negative charge on the nucleic acid molecule and condensing the nucleic acid molecule into a compact molecule, a nucleic acid associated with a lipsome (Wang, et al., PNAS 84:7851, 1987), and certain eukaryotic cells such as a producer cell, that are capable of delivering a nucleic acid molecule having one or more desirable properties to host cells in an organism. The desirable properties include the ability to express a desired substance, such as a protein, enzyme, or antibody, and/or the ability to provide a biological activity, which is where the nucleic acid molecule carried by the gene delivery vehicle is itself the active agent without requiring the expression of a desired substance. One example of such biological activity is gene therapy where the delivered nucleic acid molecule incorporates into a specified gene so as to inactivate the gene and “turn off” the product the gene was making, or to alter the translation or stability of the mRNA of the specified gene product. Gene delivery vehicle refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest or of turning off the gene of interest. The gene delivery vehicle will generally include promoter elements and may include a signal that directs polyadenylation. In addition, the gene delivery vehicle can include a sequence which is operably linked to the sequence(s) or gene(s) of interest and, when transcribed, acts as a translation initiation sequence. The gene delivery vehicle may also include a selectable marker such as Neo, SV.sup.2 Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence. Gene delivery vehicles as used within the present invention refers to recombinant vehicles, such as viral vectors (Jolly, Cancer Gen. Therapy 1:5164, 1994), nucleic acid vectors, naked DNA, oligonucleotides, cosmids, bacteria, and certain eukaryotic cells (including producer cells; see U.S. Ser. No. 08/240,030 and U.S. Ser. No. 07/800,921), that are capable of eliciting an immune response within an animal. Representative examples of such gene delivery vehicles include poliovirus (Evans et al., Nature 339:385-388, 1989; and Sabin, J. Biol. Standardization 1:115-118, 1973); rhinovirus, pox viruses, such as canary pox virus or vaccinia virus (Fisher-Hoch et a., PNAS 86:317-321, 1989; Flexner et al., Ann, N.Y. Acad. Sci. 569-86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112 4,769,330, and 5,017,487; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114, 1979); retrovirus (U.S. Pat. No. 4,777,127, GB 2,200,651, EP 0,345,242, and WO 91/02805); influenza virus (Luytjes et al., Cell 59:1107-1113, 1989; McMicheal et a., N. Engl. J. Med. 309:13-17, 1983; and Yap et al., Nature 273:238-239, 1978); adenovirus (Berkner, Biotechniques 6:616-672, 1988; Rosenfeld et al., Science 252:431-434, 1991; WO 93/9191; Kolls et al., PNAS 91:215-219, 1994; Kass-Eisler et al., PNAS 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; Guzman et all, Cir. Res. 73:1202-1207, 1993; Zabner et al., Cell 75:207-216, 1993; Li et al., Hum. Gene. Ther. 4:403-409, 1993; Caillaud et al., Eur. J. Neurosci. 5:1287-1291, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993; Jaffe et al., Nat. Genet. 1:372-378, 1992; and Levrero et al., Gene 101:195-202, 1991); parvovirus such as adeno-associated virus (Samulski et al., J. Vir. 63:3822-3828, 1989; Mendelson et al., Virol. 166:154-165, 1988; PA 7/222,684); herpes (kit, Adv. Exp. Med. Biol. 215:219-236, 1989); SV40; HIV (Poznansky, J. Virol. 65:532-536, 1991); measles (EP 0 440,219); astrovirus (Munroe, S. S. et al., J. Vir. 67:3611-3614, 1993); Semlild Forest Virus, and coronavirus, as well as other viral systems (e.g., EP 0,440,219; WO 92/06693; U.S. Pat. No. 5,166,057). In addition, viral carriers may be homologous, non-pathogenic (defective), replication competent viruses (e.g., Overbaugh et al., Science 239:906-910, 1988) that nevertheless induce cellular immune responses, including cytotoxic T-cell lymphocytes (CTL).

The term “ex vivo administration” refers to transfecting or administering a substance to a cell, for example a cell from a population of cells that are exhibiting aberrant SHIP activity, after the cell is removed from the mammal. After transfection or administration of the substance, the cell is then replaced in the mammal. Ex vivo administration can be accomplished by removing cells from a mammal, optionally selecting cells to transform, rendering the selected cells incapable of replication, transforming or treating the selected cells with a polynucleotide or other means for modulating SHIP activity, and placing the transformed or treated cells back into the mammal.

“Administration” or “administering” as uset herein refers to the process of delivering to a mammal a therapeutic agent, or a combination of therapeutic agents. The process of administration can be varied, depending on the therapeutic agent, or agents, and the desired effect. Administration can be accomplished by any means appropriate for the therapeutic agent, for example, by parenteral mucosal, pulmonary, topical, catheter-based, or oral means of delivery. Parenteral delivery can include, for example, subcutaneous, intravenous, intramuscular, intra-arterial, and injection into the tissue of an organ. Mucosal delivery can include, for example, intranasal delivery. Pulmonary delivery can include inhalation of the agent. Catheter-based delivery can include delivery by iontophoretic catheter-based delivery. Oral delivery can include delivery of an enteric coated pill, or administration of a liquid by mouth. Administration will generally also include delivery with a pharmaceutically acceptable carrier, such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome and/or a lipid. Gene therapy protocol is considered an administration in which the therapeutic agent is a polynucleotide capable of accomplishing a therapeutic goal when expressed as a transcript or a polypeptide in the mammal.

A “nucleic acid” or a “polynucleotide,” as used herein, refers to either RNA or DNA molecule that encodes a specific amino acid sequence or its complementary strand. Nucleic acid molecules may also be non-coding sequences, for example, a ribozyme, an antisense oligonucleotide, or an untranslated portion of a gene. A “coding sequence” as used herein, refers to either RNA or DNA that encodes a specific amino acid sequence, or it complementary strand. A polynucleotide may include, for example, an antisense oligonucleotide, or a ribozyme, and can also include such items as a 3′ or 5′ untranslated region of a gene, or an intron of a gene, or other region of a gene that does not make up the coding region of the gene. The DNA or RNA may be single stranded or double stranded. Synthetic nucleic acids or synthetic polynucleotides can be chemically synthesized nucleic acid sequences, and can also be modified with chemical moieties to render the molecule resistant to degradation. Synthetic nucleic acids can be ribozymes or antisense molecules, for example. Modifications to synthetic nucleic acid molecules include nucleic acid monomers or derivative or modifications thereof, including chemical moieties, such as, for example, phosphothioate modification. A polynucleotide derivative can include, for example, such polynucleotides as branched DNA (bDNA). A polynucleotide can be a synthetic or recombinant polynucleotide, and can be generated, for example, by polymerase chain reaction (PCR) amplification, or recombinant expression of complementary DNA or RNA, or by chemical synthesis.

The term “an expression control sequence” or a “regulatory sequence” refers to a sequence that is conventionally used to effect expression of a gene that encodes a polypeptide and include one or more components that affect expression, including transcription and translation signals. Such a sequence includes, for example, one or more of the following: a promoter sequence, an enhancer sequence, an upstream activation sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5′ leader sequence to optimize initiation of translation in mammalian cells, a Kozak sequence, which identifies optimal residues around initiator AUG for mammalian cells. The expression control sequence that is appropriate for expression of the present polypeptide differs depending upon the host system in which the polypeptide is to be expressed. For example, in prokaryotes, such a control sequence can include one or more of a promoter sequence, a Shine-Dalgarno sequence, a ribosomal binding site, and a transcription termination sequence. In eukaryotes, for example, such a sequence can include a promoter sequence, and a transcription termination sequence. If any necessary component of an expression control sequence is lacking in the nucleic acid molecule of the present invention, such a component can be supplied by the expression vector to effect expression. Expression control sequences suitable for use herein may be derived from a prokaryotic source, a eukaryotic source, a virus or viral vector or from a linear or circular plasmid. Further details regarding expression control sequences are provided below. An example of a regulatory sequence is the human immunodeficiency virus (“HIV-1”) promoter that is located in the U3 and R region of the HIV-1 long terminal repeat (“LTR”). Alternatively, the regulatory sequence herein can be a synthetic sequence, for example, one made by combining the UAS of one gene with the remainder of a requisite promoter from another gene, such as the GADP/ADH2 hybrid promoter.

“Hybridization” refers to the association of two nucleic acid sequences to one another by specific hydrogen bonding. Typically, one sequence can be fixed to a solid support and the other is free in solution. The two sequences are placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this binding bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of substances to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and, the stringency of the washing conditions following hybridization. See Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION (1989), Volume 2, chapter 9, pages 9.47 to 9.57. “Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 20° to 20° C. below the calculated Tm of the hybrid under study.

The term “naked DNA” refers to polynucleotide DNA for administration to a mammal for expression in the mammal or to inhibit SHIP activity. The polynucleotide can be, for example, a coding sequence, and the polynucleotide DNA can be directly or indirectly connected to an expression control sequence that can facilitate the expression of the coding sequence once the DNA is inside a cell. Alternatively, the DNA can direct production of RNA or a polypeptide that inhibits SHIP activity.

“Recombinant retroviral vector” refers to an assembly which is capable of directing the expression of a sequence(s) or gene(s) of interest. Preferably, the retroviral vector construct should include a 5′ LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3′ LTR. A wide variety of heterologous sequences may be included within the vector construct, including for example, sequences which encode a protein (e.g., cytotoxic protein, disease-associated antigen, immune accessory molecule, or replacement protein), or which are useful in and of themselves (e.g., as ribozymes or antisense sequences). Alternatively, the heterologous sequence may merely be a “stuffer” or “filler” sequence of a size sufficient to allow production of retroviral particles containing the RNA genome. Preferably, the heterologous sequence is at least 1, 2, 3, 4, 5, 6, 7 or 8 Kb in length. The retroviral vector construct may also include transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein. Optionally, the retroviral vector construct may also include selectable markers that confer resistance of recombinant retroviral vector, transduced or transfected, cells to TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more specific restriction sites and a translation termination sequence.

A “therapeutically effective amount” is that amount that will generate the desired therapeutic outcome. For example, if the therapeutic effect desired is reduction or suppression of rejection of a transplant, the therapeutically effective amount is that amount that facilitates reduction of suppression of rejection of a transplant. A therapeutically effective amount can be an amount administered in a dosage protocol that includes days or weeks of administration.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as, for example, a polypeptide, polynucleotide, small molecule (preferably a molecule having a molecular weight of less than about 10,000), peptoid, or peptide, refers to any pharmaceutically acceptable carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.

“Vector construct” refers to an assembly which is capably of directing the expression of the sequence(s) or gene(s) of interest. The vector construct can include transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post-transcriptional modification of protein. In addition, the vector construct must include a sequence which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest and acts as a translation initiation sequence. Optionally, the vector construct may also include a signal which directs polyadenylation, a selectable marker such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence. In addition, if the vector construct is place into a retrovirus, the vector construct must include a packaging signal, long terminal repeats (LTRs), and positive and negative strand primer binding sites appropriate to the retrovirus used (if these are not already present).

“Tissue-specific promoter” refers to transcriptional promoter/enhancer or locus defining elements, or other elements which control gene expression as discussed above, which are preferentially active in a limited number of tissue types. Representative example of such tissue-specific promoters include the PEP-CK promoter, HER2/neu promoter, casein promoter, IgG promoter, Chorionic Embryonic Antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, tyrosine hydroxylase promoter, albumin promoter, alphafetoprotein promoter, acetyl-cholin receptor promoter, alcohol dehydrogenase promoter, a or P globin promoters, T-cell receptor promoter, or the osteocalcin promoter.

“Mammalian cell” as used herein refers to a subset of eukaryotic cells useful in the invention as host cells, and includes human cells, and animal cells such as those from dogs, cats, cattle, horses, rabbits, mice, goats, pigs, etc. The cells used can be genetically unaltered or can be genetically altered, for example, by transformation with appropriate expression vectors, marker genes, and the like. Mammalian cells suitable for the method of the invention are any mammalian cell capable of expressing the genes of interest, or any mammalian cells that can express a cDNA library, cRNA library, genomic DNA library or any protein or polypeptide useful in the method of the invention. Mammalian cells also include cells from cell lines such as those immortalized cell lines available from the American Type Culture Collection (ATCC). Such cell lines include, for example, rat pheochromocytoma cells (PC12 cells), embryonal carcinoma cells (P19 cells), Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, as well as others. Also included are hematopoetic stem cells, neuronal stem cells such as neuronal sphere cells, and pluripotent or embryonic stem cells (ES cells).

The term “antagonist” as used herein refers to a molecule that blocks signaling, such as for example a molecule that can bind a receptor, but which does not cause a signal to be transduced by the receptor to the cell. In the case of inositol polyphosphatase 5′-phosphatases an antagonist might block signaling by binding, for example, at an SH2 domain on the molecule, or by binding, for example, so as to inhibit its phosphatase activity. In general, an antagonist of a polypeptide is an inhibitor of any biological activity of the polypeptide. A given inhibitor or agonist may target and inhibit one biological activity, while not affecting another non-target activity of the molecule.

As used herein, in one embodiment, a suitable SHIP1 inhibitor for use in the methods of the present invention can include, without limitation, the following SHIP inhibitor compound:

As used here, in other embodiments, suitable SHIP1 inhibitors for use in the methods of the present invention can include, without limitation, the SHIP inhibitor compounds of the formula (I), and pharmaceutically acceptable salts thereof, where formula (I) is as follows:

wherein:

at the 4,5 and 5,6 positions represents a single or double bond, with the proviso that the sum of double bonds present at the 4,5 and 5,6 positions is 0 or 1.

R¹ is a straight chain C₁-C₄ alkyl or C₁-C₄ haloalkyl. In one embodiment, R¹ is methyl.

R² is hydrogen, methyl, or halomethyl. In one embodiment, R² is methyl.

R³ and R¹³ (when present), are individually selected from hydrogen, substituted or unsubstituted amino, C₁-C₄ alkyl, C₁-C₄ haloalkyl, and C₁-C₄ alkenyl. In one embodiment, both R³ and R¹³ are hydrogen.

R⁴ is hydrogen, hydroxy, substituted or unsubstituted amino, alkyl, or benzyl, In one embodiment, R⁴ is hydrogen.

R⁵ represents hydrogen or an alkyl group. In one embodiment, R⁵ represents an alkyl group. In one embodiment, the alkyl group is 1, 5-dimethylhexyl. In one embodiment, R⁵ represents two hydrogen atoms or one hydrogen atom together with an alkyl group.

X¹ may be selected from the group consisting of hydrogen, hydroxy, mercapto, alkoxy, aryloxy, alkythio, and arylthio. The alkoxy, aryloxy, alkylthio, and arylthio moieties may be further substituted.

X¹ may also be selected from the group consisting of alkylcarbonamido, arylcarbonamido, aminocarbonamido, hydrazinocarbonamido, alkylsulfonamido, arylsulfonamido, aminosulfonamido, and hydrazinosulfonamido, all of which may be further substituted.

X¹ may also be selected from the group consisting of (C₁-C₄ alkyl)carbonyloxy, (C₁-C₄ alkoxy)carbonyloxy, arylcarbonyloxy, aryloxycarbonyloxy, and aminocarbonyloxy, all of which may be further substituted.

X¹ may further be selected from the group consisting of a substituted or unsubstituted amino and secondary and tertiary amino groups that include at least one C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl or heterocyclic substituent, or combinations thereof. In one embodiment, the secondary or tertiary amino group contains at least one C₁-C₄ alkyl moiety, which may be further substituted.

X¹ may further be an aminoalkyl group, amino(CH₂)_(n), where “amino” is an unsubstituted or a substituted secondary or tertiary amino ad defined above, and n is an integer from 1 to 4.

X¹ may further present a divalent oxygen moiety, ═O, or a divalent N-hydroxyamino moiety, ═NOH.

X¹ may further be an amino group, except when: R¹ and R² are each methyl; X², R³, R⁴, and R¹³ are each hydrogen; and R⁵ represents one hydrogen atom together with an alkyl group, where the alkyl group is 1, 5 dimethylhexyl alky group.

In one embodiment, X¹ cannot be hydroxy when: R³ is hydrogen, R⁴ is hydrogen, and R⁵ is one hydrogen atom together with an alkyl group.

Each X² is independently defined to represent a divalent oxo or two hydrogen atoms. In one embodiment, each X² represents two hydrogen atoms.

The compounds of the present invention, as will be appreciated by one skilled in the art, possess several potential chiral carbon atoms. As a consequence of these chiral centers, the compounds of the present invention may occur as racemates, racemic mixtures, individual diastereomers and substantially pure isomers. All asymmetric forms, individual isomers, and combinations thereof, are within the scope of the present invention.

Throughout this specification, the terms and substituents retain their definitions. Below are particular definitions of terms used here.

The term “alkyl” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon radical and includes straight or branch chain groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and higher homologs and isomers such as n-pentyl, n-hexyl, 2-methylpentyl, 1,5-dimethylhexyl, 1-methyl-4-isopropyl, hexyl and the like. Preferred alkyl groups are those of C₂₀ or below (i.e., C₁₋₂₀). A divalent radical derived from an alkane is exemplified by —CH₂CH₂CH₂CH₂—. A divalent radical derived from an alkene is exemplified by —CH═CH—CH₂—. An example of a non-limiting subset of alkyl is alkyl groups of from 1 to 10 carbon atoms (C₁₋₁₀ alkyl) e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms).

The term “alkenyl”, employed alone or in combination with other terms, means a straight chain or branched monounsaturated hydrocarbon group having the stated number of carbon atoms, such as, for example, vinyl, propenyl (allyl), crotyl, isopentenyl, and the various butenyl isomers.

Alkyl and alkenyl groups may include substituents selected from the group consisting of halo, hydroxy, cyano, mercapto, —S(C₁-C₄ alkyl), amino, substituted amino, acetamido, carboxy, trifluoromethyl, C₁-C₄ alkoxy, (C₁-C₄ alkoxy)carbonyl and aminocarbonyl.

The term “cycloalkyl” means an unsubstituted or substituted monovalent saturated cyclic hydrocarbon radical having the stated number of carbon atoms, including, various isomers of cyclopentyl and cyclohexyl. The term “cycloalkenyl” means an unsubstituted or substituted monovalent monounsaturated cyclic hydrocarbon radical having the stated number of carbon atoms, including, various isomers of cyclopentyl and cyclohexenyl. The term “cycloalkadienyl” means a monovalent diunsaturated cyclic radical having the stated number of carbon atoms, including, the various isomers of cyclopentadienyl and cyclohexadienyl. The substituents can be one or two of the same or different substituents selected from halo, hydroxy, cyano, mercapto, —S(C₁-C₄ alkyl), amino, substituted amino, acetamido, carboxy, trifluoromethyl, C₁-C₄ alkoxy, (C₁-C₄ alkoxy)carbonyl and aminocarbonyl.

The dotted lines between the 4,5 and 5,6 positions represent the presence or absence of an additional bond; that is, an unsaturation. Only one unsaturation can be present at any one time. The R¹³ shown in Formula (I) will, of course, be absent when an unsaturation is present.

The term “aryl” means an unsubstituted or substituted monovalent phenyl group. The substituents may be independently selected from halo, —OH, —SH, —S(C₁-C₄) alkyl), C₁-C₅ alkyl, C₁-C₅ alkoxy, carboxy, (C₁-C₄ alkoxy)carbonyl, aminocarbonyl, C₁-C₄ alkylaminocarbonyl, amino, acetamido, C₁-C₄ alkylamino, di(C₁-C₄ alkyl)amino or group —(CH₂)₁—R where q is 1, 2, 3, or 4 and R is hydroxy, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, amino, aminocarbonyl, C₁-C₄ alkylamino or di(C₁-C₄ alkyl)amino.

The term “benzyl” means a monovalent group in which a phenyl moiety is substituted by a methylene group. The benzyl group may include further substituents on the phenyl moiety.

The term “amino” means a group —NH₂. The term, “substituted amino” means an amino group where one or both amino hydrogens are independently replaced by a C₁-C₄ alkyl, C₂-C₄ alkenyl, C₅-C₆ cycloalkyl, C₅-C₆ cycloalkenyl, aryl, benzyl, or a group —(CH2)_(q)—R where 1 is 1, 2, 3, or 4 and R is hydroxy, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl, amino, aminocarbonyl, C₁-C₄ alkylamino or di(C₁-C₄ alkyl)amino.

The term “alkylcarbonamido” means a group (C₁-C₄ alkyl)C(O)(N)(R)—, where R represents H or C₁-C₄ alkyl. More specifically, the term “acetamido” means a group CH₃C(O)NH—. The term “arylcarbonamido” means a group (aryl)C(O)N(R)—, where R represents H or C₁-C₄ alkyl. The term “aminocarbonamido” means a group R′R″NC(O)N(R)—, where R represents H or C₁-C₄ alkyl, and R′ and R″ independently represent H, C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, or heterocyclic.

The term “alkylsulfonamido” means a group (C₁-C₄ alkyl)SO₂N(R)—, where R represents H or C₁-C₄ alkyl. The term “arylsulfonamido” means a group (aryl)SO₂N(R)—, where R represents H or C₁-C₄ alkyl. The term “aminosulfonamido” means a group R′R″NHSO₂N(R)—, where R represents H or C₁-C₄ alkyl, and R′ and R″ independently represent H, C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, or heterocyclic.

The term “alkylcarbonyloxy” means a group (C₁-C₄ alkyl)C(O)O—. The term “alkoxycarbonyloxy” means a group (C₁-C₄ alkyl)OC(O)O—. The term “arylcarbonyloxy” means a group (aryl)C(O)O—. The term “aryloxycarbonyloxy” means a group (aryl)OC(O)O—. The term “aminocarbonyloxy” means a group R′R″NC(O)O—, where R′ and R″ independently represent H, C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, or heterocyclic.

The term “halo” means chloro, bromo, fluoro or iodo. The term “mercapto” means a group —SH.

The term “heterocycle” means an unsubstituted or substituted stable 5- or 6-membered monocyclic heterocyclic ring that consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heterocyclic ring may be attached, unless otherwise stated, at any heteroatom or carbon atoms that affords a stable structure. The heterocycle may be unsubstituted or substituted with one or two substituents.

In one embodiment of the present invention, the compound of formula (I) is a compound of a formula as set forth below:

pharmaceutically acceptable salts thereof, wherein x═NR₂, NRCOR, NHCONR₂, OR, SR, OCOR, OCONR₂, or NHCNHNH₂, and wherein R═H, alkyl, cycloalkyl, aryl, or benzyl. In one embodiment, X cannot be NH₂ in compound of Formula 11. In another embodiment, X cannot be hydroxyl in the compound of Formula 20.

In some embodiments, of the invention, the compound of Formula (I) or pharmaceutically acceptable salt thereof is a compound of Formula (IA) or a pharmaceutically acceptable salt thereof:

wherein

represents a single or double bond (in particular embodiments,

represents a single bond);

R¹ and R² are individually selected from hydrogen and C₁₋₃ alkyl (e.g., methyl);

R³ is selected from hydrogen and amino;

R⁴ is selected from hydrogen, amino, and hydroxy;

R⁵ is selected from hydrogen, a divalent oxo atom, and C₁₋₁₀ alkyl (e.g., C₁-C₈ alkyl, such as, for example, C₈ alkyl, e.g., 1, 5-dimethylhexyl); and

X¹ is selected from hydrogen, amino, and hydroxy. In some embodiments, X¹ is selected from hydrogen and amino. In a particular embodiment, X¹ is amino.

In some embodiments, the compound of Formula (IA) or salt thereof is selected from a compound of Formula (IB) and (IC) below, or a pharmaceutically acceptable salt thereof:

In some embodiments, the compound of Formula (IA) or salt thereof is selected from a compound of Formula (ID)-(IO) below, or a pharmaceutically acceptable salt thereof.

In some embodiments, the inventive method comprises administering a pharmaceutically acceptable salt of a compound to any one of Formulas (I) or (IA)-(IO). In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt. In some embodiments, the pharmaceutically aacceptable salt is a salt of a compound wherein X¹ is amino (for example, a hydrochloride salt of such a compound, e.g., the pharmaceutically acceptable salt may be a compound having NH₃Cl at the X¹ position).

In some non-limiting embodiments, the inventive method comprises administering a compound of Formula (I) (or any sub-genus thereof) as described herein, or a pharmaceutically acceptable salt thereof, with the proviso that if X¹ is hydroxy, then R⁴ is a hydrogen, substituted or unsubstituted amino, C₁-C₄ alkyl, or benzyl.

In some non-limiting embodiments, the inventive method comprises administering a compound of Formula (I) (or any sub-genus thereof) as described herein, or a pharmaceutically acceptable salt thereof, with the proviso that if X¹ is hydroxy, at least one of R³ and R⁴ is other than hydrogen.

In some non-limiting embodiments, the inventive method comprises administering a compound of Formula (I) (or any sub-genus thereof) as described herein, or a pharmaceutically acceptable salt thereof, with the proviso that if X¹ is hydroxy, R⁵ is not an alkyl group.

In some embodiments, the inventive method comprises administering a compound selected from one of the following:

The “SHIP inhibitor compounds” of the present invention are also referred to herein as “SHIP inhibitors,” “SHIP1 inhibitors,” “SHIP1 inhibitor compounds,” “pan-SHIP1/2 inhibitors,” and the like. In one embodiment, the SHIP inhibitor compounds of the present invention are selective inhibitors of SHIP1.

As used herein, suitable pan-SHIP1/2 inhibitors for use in the methods of the present invention can include, without limitation, the pan-SHIP1/2 inhibitor compounds as follows:

Various aspects and embodiments of the present invention as they relate to the SHIP1 and pan-SHIP1/2 inhibitors are further described in the Examples and the associated figures and tables provided herewith in connection with the Examples.

As used herein, in other embodiments, suitable SHIP1 inhibitors for use in the methods of the present invention can include, without limitation, small interfering RNAs (siRNAs) or microRNAs (miRNAs) that are effective to inhibit SHIP1 via RNA interference (RNAi) (post transcriptional gene silencing).

RNAi technology provides an efficient means for blocking expression of a specific gene. RNAi technology takes advantage of the cell's natural machinery, facilitated by short interfering RNA molecules, to effectively knock down expression of a gene of interest. There are several way to induce RNAi, synthetic molecules, siRNA, miRNA, RNAi vectors, and in vitro dicing.

RNAi can be used to inhibit the SHIP1 genes, such as by creating siRNAs or miRNAs having the appropriate sequence and delivering them to the cells in which inhibition of the SHIP1 gene is desired. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral vector systems similar to those suggested for gene therapy. Once developed, these delivery methods can be used for the purposes of the present invention. RNAi inducing agents can also be delivered using bacteria, retroviruses, DNA viruses, lipidoids and amphoteric liposomes.

General rules for selecting siRNA targets on mRNA sequences include, for example, the following (www.maiweb.com/RNAi/siRNA_Design/): (i) Targets should be located 50-100 nt downstream of the start codon (ATG): (ii) Search for sequence motif AA(₁₉)TT or NA(N₂₁), or NAR(N₁₇)YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U); (iii) Target sequences should have a G+C content between 35-60%; (iv) Avoid stretches of 4 or more nucleotide repeats; (v) Avoid 5′URT and 3′UTR, although siRNAs targeting UTRs have been shown to successfully induce gene silencing; and (vi) Avoid sequences that share a certain degree of homology with other related or unrelated genes.

Selecting targets for miRNA: In animals, the tendency of miRNAs to bind their mRNA targets with imperfect sequence homology poses considerable challenges with target prediction. In animals, target sites are often only partially complementary to their miRNAs and are mostly located in the 3′UTR of target genes. Several computational approaches have been developed to facilitate experimental design and predicting miRNA targets. In general, computational target prediction identifies potential binding sites according to base-pairing rules and cross species conservation conditions.

The dosage form of the SHIP inhibitor of the present invention may be a liquid solution ready for use or intended for dilution with a preservation solution. Alternatively, the dosage form may be lyophilized or power filled prior to reconstitution with a preservation solution. The lyophilized substance may contain, if suitable, conventional excipients.

Other than in the operating examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for amounts of materials, times and temperatures of reaction, ratios of amounts, values for molecular weight (whether number average molecular weight (“M_(n)”) or weight average molecular weight (“M_(w)”), and others in the following portion of the specification may be read as if prefaced by the word “about” even thought the term “about” may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.

As used herein, the term “pretreating” or (or “pretreatment”) is intended to mean that a first treatment is administered prior to, or in conjunction with, a second treatment. In other words, the pretreatment may be performed before another, later treatment, thus allowing the pretreatment time to take effect. Alternatively, the pretreatment may be performed or administered simultaneously with a second treatment without a temporal delay. Advantageously, a pretreatment is administered prior to a second treatment.

The term “administration” and variants thereof (e.g., “administering” a compound) in reference to a compound of the invention can also mean introducing the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), “administration” and it variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.

As used here, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly and indirectly, from combination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein can also means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

A “subject in need of treatment” is a mammal with a bone-loss condition.

A “pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

A “safe and effective amount” refers to the quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ration when used in the manner of this invention.

A “pharmaceutically acceptable carrier” can also refer to a carrier, such as a solvent, suspending agent or vehicle, for delivering the compound or compounds in question to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutical carrier. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instance where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods. See, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and Applications (Innis, et a. 1990. Academic Press, San Diego, Calif.), McPherson et al., PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney, 1987. Liss, Inc, New York, N.Y.), and Gene Transfer and Expression Protocols, pp. 109-128, ed; E. J. Murray, The Humana Press Inco., Clifton, N.J.).

3β-Amino-5α-Androstane Hydrochloride (K118)

As used herein, in one embodiment, a suitable SHIP inhibitor for use in the methods of the present invention can include, without limitation, the following SHIP inhibitor compound of Formula 28, wherein X═NH2 or NH₃Cl, as well as an derivatives or analogs thereof:

More particularly, the compound described here as “K118” refers to the SHIP inhibitor compound of Formula 28 wherein X is NH3Cl. K118 is also referred to here as 3β-amino-5α-androstane hydrochloride. Aspects of K118 are further described in Example 20, Example 21, and the figures associated with the relevant Examples. For example, as described in Example 21, K118 can be effective as a SHIP inhibitor to prevent or reduce obesity without negatively impacting bone density. K118 is a water-soluble derivative of 3AC and has comparable SHIP1 inhibitory activity. Because K118 is water-soluble, it can be used for pharmacological targeting of SHIP1. K118 can also be described as being a pan-SHIP1/2 type of inhibitor.

Various analogs of K118 can include, without limitation, the compounds identified herein as Formula 11, Formula 14, Formula 17, Formula 20, Formula 23, Formula 24, Formula 25, Formula 31, Formula 32, Formula 33, Formula 34, Formula 35, Formula 36, and Formula 37, wherein X═NH2 or NH3Cl.

Provided below are more particular terms and aspects regarding various embodiments for the use of K118 as a therapeutic composition, although the use of K118 is not meant to be limited by the terms and aspects described below. Further, as used herein, reference to K118 is also meant to relate to the derivatives, analogs, and any variations of K118.

An “effective amount” of K118, and pharmaceutically acceptable salts or derivatives thereof, may be in a dosing range of from about 0.05 mg/kg to about 150 mg/kg and particularly in a dosing range of from about 0.1 mg/kg to about 100 mg/kg. More particularly, the dosing range can be from 0.08 mg/kg to 140 mg/kg, from 0.1 mg/kg to 130 mg/kg, from 0.1 mg/kg to 120 mg/kg, from 0.1 mg/kg to 110 mg/kg, from 0.1 mg/kg to 110 mg/kg, from 0.5 mg/kg to 100 mg/kg, from 1 mg/kg to 100 mg/kg, from 10 mg/kg to 80 mg/kg, from 20 mg/kg to 70 mg/kg, from 20 mg/kg to 60 mg/kg, from 20 mg/kg to 50 mg/kg, from 20 mg/kg to 40 mg/kg, and from 20 mg/kg to 30 mg/kg.

A “pharmaceutically acceptable derivative” means any non-toxic salt, ester, salt of an ester or other derivative of a compound of this invention that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitory active metabolite or residue thereof.

In one embodiment of the present invention, K118 is administered at a dose from 0.05 mg/kg to 150 mg/kg or more particularly at a dose from 0.1 mg/kg to 100 mg/kg once a day every other day, three times a week, twice a week, once a week, etc. In another embodiment, K118 is administered at a dose from 0.08 mg/kg to 140 mg/kg, from 0.1 mg/kg to 130 mg/kg, from 0.1 mg/kg to 120 mg/kg, from 0.1 mg/kg to 110 mg/kg, from 0.1 mg/kg to 110 mg/kg, from 0.5 mg/kg to 100 mg/kg, from 1 mg/kg to 100 mg/kg, from 10 mg/kg to 80 mg/kg, from 20 mg/kg to 70 mg/kg, from 20 mg/kg to 60 mg/kg, from 20 mg/kg to 50 mg/kg, from 20 mg/kg to 40 mg/kg, and from 20 mg/kg to 30 mg/kg once a day, every other day, three times a week, twice a week, once a week, etc.

The term “pharmaceutically acceptable” means that a compound or combination of compounds is sufficiently compatible with the other ingredients of a formulation, and not deleterious to the patient up to those levels acceptable by the industry standards.

Therefore, K118 may be formulated into various pharmaceutical forms for administration purposes. As appropriate compositions there may be cited all compositions usually employed for systemically administering drugs. To prepare the pharmaceutical compositions of this invention, an effective amount of K118 as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the manner of preparation desired for administration.

These pharmaceutical compositions are desirable in unitary dosage form suitable, particularly, for administration orally, rectally, intraperitoneally, transdermally, intradermally, topically, by inhalation, nasally, buccally, vaginally, via an implanted reservoir or by parenteral routes. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants or vehicles that may be used in the compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

In one embodiment of the present invention, K118 is administered orally. K118 can be administered by the oral route in solid dosage forms, such as tablets, capsules, and powders, or in liquid dosage forms, such as elixirs, syrups, suspensions, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The pharmaceutical compositions of this invention can also be administered parenterally, in sterile liquid dosage forms.

In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Liquids dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are obviously employed.

K118 may as well be administered in oral dosage forms such as the ones described in U.S. Pat. No. 7,182,958, as a free drug in admixture with a diluent, a lubricant, a hydrophilic binder selected from the group consisting of a cellulose derivative, povidone, and a mixture thereof, a disintegrant selected from the group consisting of crospovidone, croscarmellose sodium, and a mixture thereof, and, optionally, microcrystalline cellulose and/or a wetting agent. Optionally, the formulation additionally comprises a second diluent.

K118 may as well be administered as a coprecipitate preparation with a polymer, as disclosed in U.S. Pat. No. 5,985,326, wherein the polymer is for example hydroxypropyl methylcellulose phthalate. This coprecipitate preparation is prepared, then milled, mixed with excipients, and compressed into tablets for oral administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

In preliminary studies, K118 was shown to induce increases in immunoregulatory cells when delivered orally at 10 mg/kg. A significant increase was observed in the frequency of myeloid derived suppressor cells (MDSC) expressing both Gr1 and Mac1 cell markers. This was observed in the spleen of treated mice. Significant increases were also observed in the frequency of “natural” T regulatory cells (nTreg), characterized by expression of CD4⁺CD25⁻FoxP3⁺, in both the spleen and in the mesenteric lymph node (mLN). Finally, it was observed a trend for increased neutrophil numbers, as is observed with intraperitonial injection of SHIP1 inhibitor 3AC. K118 was administered in water, and in wt C57BL/6 micc.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelating capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Provided compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coating, release controlling coatings and other coating well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, and eye drops are also contemplated as being within the scope of this invention. Additionally, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrance or by dispersing the compound in a polymer matrix or gel.

Also, in certain embodiments, free K118 drug is preferred in particulate form, and wherein at 90% of the particles have a particle size of less than about 40 microns, and preferably less than 30 microns. Highly preferred particulate forms of the compound (I) have at least 90% of the particles less than 25 microns in size. Most preferred forms of the free compound (I) are those wherein 90% of the particles are less than 10 microns in size, as described and prepared in U.S. Pat. No. 6,821,975.

Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. Injectable solution, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. These latter suitable additives may be anti-oxidants, preservatives, stabilizing agents, emulsifiers, salts for influencing the osmotic pressure, and/or buffer substances.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a provided compound, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled.

Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

In one embodiment of the present invention, K118 is administered transdermally. In one embodiment of the present invention, K118 is administered topically.

As appropriate topical or transdermal compositions there may be cited for example gels, jellies, creams, pastes, emulsions, dispersions, ointments, films, sponges, foams, aerosols, powders, implants, patches. In the compositions suitable for topical cutaneous administration, the carrier optionally comprises a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various way, e.g., as a cream or gel.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Pharmaceutically acceptable compositions provided herein may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

Pharmaceutically acceptable compositions provided herein may be formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this disclosure are administered without food. In other embodiments, pharmaceutically acceptable compositions of this disclosure are administered with food.

The amount of provided compounds that may be combined with carrier materials to produce a composition in a single dosage from will vary depending upon the patient to be treated and the particular mode of administration. Provided compositions may be formulate such that a dosage of between 0.01-150 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, and segregated multiples thereof.

Notwithstanding the effective amounts and doses indicated above, still the dose of K118, its pharmaceutically acceptable salts and solvates thereof to be administered will depend on the individual case and, as customary, is to be adapted to the conditions of the individual case for an optimum effect. Thus it depends, of course, on the frequency of administration and on the potency and duration of action of the compound employed in each case for therapy or phophylaxis but also on the nature and severity of the disease and symptoms, and on the sex, age, weight co-medication and individual responsiveness of the subject to be treated and on whether the therapy is acute or prophylactic. Doses may be adapted in function of weight and for paediatric applications. Daily doses may be administered q.d. or in multiple quantities such as b.i.d., t.i.d. or q.i.d. Alternatively, doses may be administered every other day, every three, every four, every five, every six, every seven day, every other week, every month.

In one embodiment, the SHIP1 or pan-SHIP1/2 inhibitor is injected intraperitoneally at between about 10 mg/kg and 80 mg/kg of body weight.

In another embodiment, the SHIP1 or pan-SHIP1/2 inhibitor is injected intraperitoneally at between about 10 mg/kg and 80 mg/kg of body weight.

In another embodiment, the administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor results in an increase in number of hematopoietic stem cells and/or mesenchymal stem cells in the subject.

In another embodiment, the administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor results in an increase in number of neutrophils in the patient.

In another embodiment, the subject is suffering from a bacterial, fungal, or parasitic infection, a cancer, chemotherapy- or radiation-induced neutropenia, radiation-induced myeloablation, the effects of immunosuppressive drugs, or declining stem cell function associated with genetic, environmental or age-related factors.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a SHIP inhibitor compound, including, without limitation, a SHIP1 inhibitor and/or a pan-SHIP1/2 inhibitor compound as described herein, or a pharmaceutically acceptable salt thereof.

EXAMPLES

The following examples are intended to illustrate particular embodiments of the present invention, but are by no means intended to limit the scope of the present invention.

Example 1 Coordinate Expansion of Murine Hematopoietic and Mesenchymal Stem Cell Compartments of SHIPi

It was hypothesized that transient inhibition of SHIP1 in vivo (SHIPi) might offer an approach to expand HSC without adversely impacting their function or the survival of the host. Previously we showed that transient in vivo inhibition of SHIP1 with the small molecule inhibitor 3AC replicates several immune and hematologic phenotypes present in germline SHIP1^(−/−) mice.¹³ However, SHIPi treatment does not induce the development of pulmonary and gastrointestinal inflammation that compromises the viability of SHIP1^(−/−) mice.^(13, 14) Here we describe the in vivo use of SHIPi to expand both the MSC and HSC compartments in adult hosts.

Materials and Methods Used for Expansion of HSC and MSC Compartments Using SHIPi

Mice: HSC phenotype, cell-cycle progression, G-CSF neutralization, and CXCR4 MFI determination by FACS analysis was performed on whole bone marrow (WBM) cells harvested from a tibia and femur pair obtained from SHIPi and vehicle treated 8-12 week old male C56BL/6NTac mice (Taconic). Generation of the CD45.1×CD45.2 strain utilized in the CRU assay were obtained by intercrossing C56BL6/J (CD45.2) and B6.SJL (CD45.1), as previously described.⁵ The SUNY Upstate Medical University Committee for Humane Use of Animals approved all animals experiments.

SHIPi Treatment of Mice

In vivo administration of 3AC was previously described.¹⁴ Briefly, 3AC is resuspended as an emulsion in 0.3% Klucel/H20. Mice were treated via intraperitoneal injection every 24 hrs for 5 days at a final concentration of 26.5 mg/kg. Analysis and transplants were performs on day 6. The 3AC derivative K118 (Soluble SHIPi) was suspended in Pure H₂O and heated at 100° C. for ˜15 min to allow the compound to enter solution. The delivery method was the same as with 3AC, however, the mice were treated for 5 days at a final concentration of 10 mg/kg. Vehicle cohorts for 3AC were treated with 0.3% Klucel/H20 and K118 were treated with pure H₂O.

Cell Isolation for HSC Assays

Cell isolation was performed according to method detailed in Hazen et al.⁶ Briefly, WBM was flushed from both femurs and tibias per mouse with a 25-gauge needle, red blood cell lysed (eBioscience), and resuspended in staining media comprised of Hanks Buffered Saline Solution (HBSS), 2% heat inactivated fetal bovine serum (FBS), and 10 mM, HEPES (Sigma). The cells harvested for the CRU assay were flushed, red blood cell lysed, washed, and resuspended in sterile phosphate buffered saline (PBS). Peripheral blood mononuclear cells (PBMC) were collected via submanibular bleed in EDTA Mirovette tubes (SARSTEDT).

Flow Cytometry Analysis

All antibodies were from BD Biosciences or eBioscience. All WBM, PBMC and BM derived MSC cell populations were Fc blocked on ice for 10 min prior to antibody staining. Global derived MSC cell populations were Fc blocked on ice for 10 min prior to antibody staining. Global reconstitution was determined by staining PBMC with CD45.1-PeCy7 and CD45.2-APC. Multi-lineage repopulation was determined by staining PBMC with CD3-FITC (145-2C11), CD19-PerCP5.5 (ID3), Mac1-PE (M1/70), Gr1-Alexa700 (RB6-8C5) and NK1.1-APC−Cy7 (PK136). The Lin-cKit+Scal+CD34-Flk2- (LSKF34-) phenotype was utilized to determine frequency of LT-HSC, ST-HSC, and MPP; the stain was comprised of Lin-PerCP 5,5, cKit-APC (2B8), Sca1-PeCy7 (D7), CD34-FITC (RAM34), Flk2-PE (A2FI0). LSKF-CXCR4 stain was used to determine surface expression of the homing marker CXCR4. All cells were washed twice with staining media, resuspended in staining media plus DAPI for dead cell exclusion, and then analyzed on a flow cytometer LSCRII (BD Biosciences). All flow cytometry data was analyzed using FlowJo (9,4.3) software.

Cell Cycle Analysis

WBM cells were stained with LSKCD34 and fixed in BD Cytofix/Cytoperm buffer for 30 minutes on ice. Fixed cells were stained with 10 μM Hoechst 33342 (HO; Sigma-Aldrich) for 45 min at 37° C. The cells were then incubated with 5 μM Pyronin Y (PY; Sigma-Aldrich) in HBSS medium with 2% FBS in the presence of 50 μM verapamil (Sigma-Aldrich) for 30 minutes at 37° C., washed, and immediately analyzed by UV laser-equipped BD LSRII analyzer.

CRU Assay

CD45.1⁺45.2⁺-recipient mice were given a split dose of 550 Rads (x-ray source, RS2000-RAD source technologies, inc.) 4 hours apart for a lethal total dose of 1100 Rads. WBM (5×10⁵) cells from 3AC (CD45.2) treated mice were competed with WBM cells from vehicle (CD45.1) treated mice and injected retro-orbitally into CD45.1⁺45.2⁺ hosts. PBMC multi-lineage reconstitution was evaluated every month post-transplant for 4 months. Transplanted mice were euthanized and serially transplanted to determine self-renewal capacity. Repopulating Units (RU) determination was previously described.¹⁵ Briefly, RU=(C×CD45.2 repopulation %)/(100=CD45.2 repopulation %) where C=10⁵ competitor cells.⁶

Calculation of Absolute RU

Normal Repopulating units (RU) are calculated based on total number of competitor cells transplanted. Due to the expansion in the bone marrow compartment of SHIPi treated mice, we calculated the absolute RU numbers based on RU output determined by CRU assay and total BM cellularity. Total BM cellularity was determined via hemacytometer (BioRAD, TC20 cell counter) based on a total BM cells obtained from an intact set of femurs and tibias for each mouse in the study. Absolute RU=(Total BM cellularity/5×10⁵)×RU.

In Vivo MSC Immunophenotyping by Flow Cytometry

PDGFRα⁺CD51⁺ MSC were detected using methods and reagents as described by Iyer et al.⁸ Femurs, tibias, humeri, ulnas, radii, and pelvis were dissected; the bone fragments were cleaned, cut into tiny pieces, and then crushed using a mortar and pestle. The bone chips were then washed several times in HBSS supplemented with 2% heat-inactivated FBS (HBSS/2% HI-FBS) and HBSS only, and then spun at 350×g for 5 mins. The pellet was re-suspended in 3 mg/mL type 1 collagenase (Worthington) and 15 μg/mL DNAse (Sigma, St. Louis, Mo.) dissolved in HBSS and incubated at 37° C. for 30 min and 110 rpm, this step was repeated twice. Following incubation, mononuclear cells were obtained using a Ficoll gradient (Histopaque-1119 and Histopaque-1077; Sigma, St. Louis, Mo.), and were then used for further analysis. Cells were stained with antibodies as described in Iyer et al.⁸

Colony Forming Unit Assay

Primary while BM cells (adherent+non-adherent) were plated in 60-mm plates (triplicates, 3×10⁶ cells per plate) in CFU-F growth media: DMEM, Mesencult Serum (Stem Cell Technologies) with 1% penicillin-streptomycin-glutamine. Cells were cultured for approximately 7 days, after which cells were fixed with 70% ethanol and stained for alkaline phosphatase activity using the Leukocyte Alkaline Phosphatase Kit (Sigma) and counter-stained with neutral red (Sigma), Colonies (ALP⁺ cells/colonies) were counted using the Software program “Image J” (National Institutes of Health Research Service Branch).

G-CSF Elisa

Peripheral blood was collected in serum separation microvette tubes (SARSTEDT). Tubes were kept at room temperature for 2 hrs, and the spun at 2000×g for 20 min as per manufactures recommendation. Serum levels of G-CSF in treated mice were determined using a Mouse G-CSF Quantikine Elisa kit (R&D systems).

In Vivo Neutralization of G-CSF

The neutralization experiments were performed with murine anti-G-CSF (MAB414) and isotype control Ab (MAB005) were purchased from R&D systems. Normal SHIPi injection protocols were performed with the following exception, the SHIPi treatment groups were injected with 10 mg of anti-G-CSF or with 10 mg of isotype control Ab directly after 3AC injection on each of day of SHIPi treatment, as performed in Shojaei et al.¹⁶ Cells were harvested and cytometry analysis was performed as mentioned previously.

Lymphocyte and WBC Recovery

We utilized a radiation-induced hematological myeblation to create our bone marrow failure model. C56BL/6J mice (8-12 week) (The Jackson Laboratory, Bar Harbor, Me.) were sub-lethally irradiated at 550 Rads. 3AC and vehicle were administered daily for 7 days post irradiation via IP injection. PB was collected by submadibular bleeding on day 8, 15, 22 and 32 and analyzed for WBC and lymphocyte counts [Thousands per microliter (K/uL)] on a Hemavet 950S (Drew Scientific, Waterbury, Conn., USA) analysis.

Statistical Analysis

Statistical analyses were performed using Prism (GraphPad, San Diego, Calif.), where p>0.05 ns, p<0.05 *, p<0.01 **, p<0.001 ***, p<0.0001 ****. All experiments are representative of two independent comparisons of SHIPi vs vehicle treated mice with n=5 for each group. Total statistical n for all experiments n=10.

Results Related to Expansion of HSC and MSC Compartments Using SHIPi SHIPi Promotes Expansion of the HSC Compartment Without Compromising Homing:

SHIP1 deficiency leads to significant increases in phenotypic HSC numbers.⁵ However, SHIP1^(−/−) HSC show reduced functional capacity related to poor BM homing.^(5, 7) Germline mutation or systemic deletion of SHIP1 in the adult is accompanied by sever inflammatory disease that adversely impacts normal cell and tissue function, including down-regulation of CXCR4 on HSC which is critical for their niche homing and retention.^(5, 6) Consistent with this inflammation-related cell-extrinsic effect on HSC, CXCR4 expression is not lost upon SHIP1 deletion in MxCreSHIP^(flox/flox) HSC present in WT hosts where inflammatory disease is absent.⁶ We hypothesized then that transient inhibition of SHIP1 might enable the expansion of functional adult HSC without inducing the inflammatory disease observed with irreversible genetic ablation of SHIP1. We first examine whether SHIPi promotes cycling and expansion of the HSC compartment. C56BL6 mice were treated with the SHIP inhibitor 3AC for 5 days to determine if HSC numbers are increased in the bone marrow (BM). We find that SHIPi treated hosts exhibit a significant increase in bone marrow cellularity (BMC). (FIG. 1A) Consistent with the increased frequency of transitory amplifying cells, Lin-Sca1+cKit+ (LSK), observed in SHIP^(−/−) mice, we find that SHIPi significantly increases both the frequency and absolute number of LSK cells (FIG. 1B, FIG. 1C, & FIG. 7A) Importantly, SHIPi also increases the frequency of long-term HSC (LT-HSC) in BM as defined by the LSKFlk2⁻CD⁻ phenotype, short-term HSC (ST-HSC) as defined by the LSKFlk2⁻CD⁺ phenotype and multi-potent progenitors (MPP) as defined by the LSKFlk2⁻CD⁺ phenotype.¹⁷ (FIGS. 7B-7D) The increased frequency of LT-HSC, combined with the increased BM cellularity induced by SHIPi, results in a multiplicative expansion of 3-4 fold in BM of cells with a primitive HSC phenotype. (FIGS. 1D-1G) Analysis of cell cycle distribution in the LT-HSC compartment of SHIPi treated mice showed that a significantly greater frequency of these cells are in the S and G2 phases of the cell cycle, indicating expansion of HSC is due largely to increased cycling of the HSC compartment. (FIGS. 1H and 1I) A similar expansion of cells with an HSC phenotype was observed in SHIP1^(−/−) mice,⁵ but the expanded HSC is due largely to increased defective repopulating ability due to a homing defect correlated with loss of CXCR4 surface expression.⁵ However, SHIPi does not cause a reduction in CXCR4 surface expression on the expanded HSC pool. (FIGS. 7E and 7F) This suggested that unlike the expanded HSC in germline SHIP1^(−/−) where CXCR4 is down regulated, SHIPi expanded HSC demonstrate potential for normal reconstituting function following intravenous transplant.

SHIPi Induced HSC Retain Normal Long-Term Multi-Lineage Repopulation and Self-Renewal Capabilities

To determine if the phenotypic increase in LT-HSC induced by SHIPi corresponds to a functional increase in HSC activity, we measured the frequency and number of competitive repopulating units (CRU)¹⁸ in the BM of SHIPi mice vs. vehicle treated controls. To accomplish this, we performed CRU assays where whole BM cells (WBM) from SHIPi treated CD45.2 mice were transplanted in competition with an equal number of BM cells from vehicle treated CD45.1 congenic donors into CD45.1⁺45.2⁺ heterozygous hosts. Global and lineage-specific reconstitution by the competing donor BM cells was then assessed in the peripheral blood (PB) of CD45.1⁺45.2⁺ host at monthly intervals for four consecutive months post-transplant. Global blood cell reconstitution by BM cells from SHIPi donors was significantly greater than that of an equal number of competitor cells from BM of vehicle treated donors at all time points analyzed. (FIG. 2A, FIGS. 8A-8D) Moreover, HSC from SHIPi conditioned host's reconstitute a greater proportion of all major blood cell lineages analyzed, including all three lymphoid lineages (B, T, NK) as well as the myeloid lineage (Mac1Gr1), during the entire post-transplant period. (FIG. 2B, FIG. 8) Consistent with increased global repopulation SHIPi also increases CRU frequency (FIG. 2C) and, after taking into account the increase in total BM cellularity, it induces a ˜6-fold increase in cells with HSC capacity based on comparisons of absolute CRU numbers in SHIPi and vehicle treated BM donors. (FIG. 2D) We conclude then that SHIPi promotes cycling and expansion of the HSC compartment in a manner that does not alter or impair their long-term multi-lineage repopulating potential.

The increased cycling of HSC that is required to support expansion of the HSC compartment could potentially harm the self-renewal capacity of SHIPi expanded HSC. We sought to determine if SHIPi damages HSC self-renewal by performing a secondary CRU assay, where primary CRU assay BM was harvested and transplanted into secondary CD45.1⁺45.2⁺ heterozygous hosts. One month after serial transfer, PBMC were analyzed for both global (FIG. 2E) and lineage-specific secondary reconstitution. (FIG. 2F) As in the primary CRU hosts, we did not observe a significant bias or impairment in lineage output of SHIPi BM derived HSC following serial transfer to secondary hosts. In fact, repopulation of all major lineages by HSC derived from SHIPi BM donors continued to be significantly higher versus competing vehicle BM. (FIG. 2F) SHIPi expanded HSC therefore show no evidence of senescence or development bias that can occur with increased cyclic activity.^(19, 20)

SHIPi Results in the Expansion of HSC Niche Supporting Cells

The BM HSC niche is comprised of a multitude of cells that support or maintain primitive HSC. Although there is significant cellular complexity in this niche and potentially more than one HSC niche, mesenchymal stem cells (MSC) are a central player in all niches that support primitive HSC subsets.²¹ SHIP1 is expressed by BM niche cells including MSC, and moreover has a functional role in HSC support.^(6, 8) We reasoned that the profound HSC compartment increase induced by SHIPi that occurs without apparent loss of functional capacity would be unlikely to develop without a coordinate expansion of the MSC. Significant advances in the prospective identification of murine MSC over the last several years has enabled their multi-lineage potential and self-renewal capacity to be demonstrated at a clonal level.²¹⁻²⁴ Thus, we quantified PDGFRα⁺51⁺CD31⁻Lin⁻CD45⁻ cells (Pα⁺51⁺MSC)²³ in the BM of SHIPi or vehicle cohorts and observed an approximate doubling of the Pα⁺51⁻ MSC compartment in SHIPi treated mice. (FIGS. 3A-3B) Consistent with these findings, the MSC pool is also expanded in mice where SHIP1 is selectively deleted, indicating that SHIP1 also limits MSC compartment size in vivo.⁸ Taken together, these findings strongly suggest that SHIPi dynamically changes the BM microenvironment by expanding a key niche cell component and this contributes to the profound expansion of functional HSC induced by SHIPi.

SHIPi Induces G-CSF Production and Drives the Expansion of Bothe HSC and MSC Compartments

Our previous work showed that not only was the cellular composition of the BM niche altered by SHIP1 deficiency, but the production of soluble mediators from the BM that impact HSC self-renewal and maintenance were altered as well.⁶ The most prominent alteration that we observed was an approximate five-fold increase in G-CSF production in SHIP1^(−/−) mice.⁵ We considered that SHIP1 might limit HSC and MSC expansion in vivo by preventing over-expression of G-CSF and thus limit this growth factor's impact on cell signaling in the HSC and/or MSC compartments. As anticipated from these genetic studies, SHIPi treatment of mice also increases G-CSF production in vivo to a concentration comparable to that observed in SHIP1^(−/−) mice. (FIG. 4A) To determine if induction of G-CSF is responsible for the HSC and MSC expansion mediated by SHIPi, we neutralized G-CSF in vivo by antibody administration. Neutralization of G-CSF prevented the increase in BM cellularity (FIG. 4B) and the expansion of both the LT-HSC (FIG. 4C) and MSC (FIG. 4D) compartments in SHIPi treated mice, but did not adversely impact HSC and MSC numbers in vehicle controls. Thus, SHIPi primarily mediates its effect on stem cell expansion via induction of G-CSF, a growth factor that promotes cycling of both HSC and MSC.^(25, 26) In addition, SHIPi may also increase the proliferation of HSC and MSC by blocking SHIP1 function at the G-CSF receptor (G-CSFR) as SHIP1 is recruited to G-CSFR, where it antagonizes proliferation driven by PI3K signaling.^(27, 28)

SHIPi Promotes Improved Pan-Hematolymphoid Recovery in an Induced Bone Marrow Failure Model

The expansion observed in both the HSC and MSC compartments could have a significant impact in a clinical setting, particularly in bone marrow failure, where the function of one or both stem cell populations may be compromised. To determine if SHIPi treatment could potentially expand a stressed or depleted HSC compartment, we utilized a radiation induced bone marrow failure model.²⁹ We previously found that SHIPi treatment post radiation-induced myeloablation promoted a more rapid recovery of both neutrophils and platelet numbers.¹³ We further analyzed hematopoietic recovery in this model and found that SHIPi also promotes significantly improved recovery of white blood cell (WBC) and lymphocyte numbers after radiation-induced myeloablation. (FIG. 5A-5B) These observations are consistent with improved pan-hematolymphoid recovery promoted by increased HSC function in SHIPi treated hosts.

A Water-Soluble SHIP Inhibitor Compound for Stem Cell Expansion

The above studies suggest significant therapeutic potential of SHIPi for in vivo expansion of adult stem cell populations. However, SHIPi utilizing the 3AC compound might pose practical limitations due it its poor aqueous solubility.³⁰ We recently identified and H₂0 soluble derivative of 3AC, K118, which based on fluorescent polarization (FP) assays for SHIP1 activity, has equal potency for inhibition of SHIP1 activity in vitro. (FIG. 6A) We sought to determine if this water-soluble K118 SHIPi compound has the capacity to increase G-CSF production within the same five-day injection period as 3AC. We observed a significant increase in G-CSF production in the K118 group as compared to vehicle (H₂0) controls. (FIG. 6B) Similar increases were observed in BM cellularity (data not shown) as well as significant increases in all three developmental (LT-HSC, ST-HSC, and MPP) stages of the HSC compartment. (FIGS. 6C-6D) Primary CRU assays performed with WBM from K118 treated (CD45.2) and H₂0 treated (CD45.1) mice show a significant increase in CRU activity (FIG. 6E), which corresponds to the observed CRU increase in 3AC treated recipients. As demonstrated by 3AC treatment, the significant increase in BM cellularity induced by K118 also leads to a significant increase in absolute CRU numbers. (FIG. 6F) Finally, a significant expansion of the MSC numbers was observed in the BM of K118 treated mice, similar to the expansion triggered by 3AC SHIPi treatment. We also performed a CFU-F assay to determine whether the phenotypic increase observed by flow cytometry corresponded to a functional MSC increase. Indeed, BM of K118 treated hosts shows significantly increased frequency of CFU-F as compared to H₂0 controls. Thus, SHIP1 can be targeted in vivo with a water-soluble inhibitor that is better suited for further therapeutic development.

Discussion of Results Related to Expansion of HSC and MSC Compartments Using SHIPi

Here we describe the development of SHIP inhibitory approaches (SHIPi) that can induce significant expansions of two key adult stem cell populations, the HSC and the MSC. This effect appears to be largely mediated by induction of G-CSF that in turn promotes expansion of HSC and MSC in vivo. Importantly, expansion of the HSC compartment does not appear to compromise or limit self-renewal potential of these cells. Of relevance to further pharmaceutical development for SHIPi approaches, we show that induction of G-CSF and expansion of stem cells in vivo can be achieved with a SHIPi molecule that has aqueous solubility. Based on these studies and our analysis of SHIPi in the radiation-induced myeloablation recovery model, this methodology is a feasible means to achieve in vivo expansion of stem cell populations with potential clinical applicability in a number of therapeutic areas.

Intracellular downstream activation of molecules with essential roles in cellular expansion and survival has distinguished the inositol phospholipid-signaling cascade, and the genes that encompass it, as tractable therapeutic targets.^(8, 13, 14, 31) At the center of the pathway lies PI3K, whose enzymatic function creates plasma membrane docking sites for recruitment of AKT allowing for its subsequent activation and enhanced proliferation and survival of hematopoietic stem and progenitor cells.^(1, 32) PI3K signaling is attenuated by the 3′ and 5′ inositol phosphatases, PTEN and SHIP1, which are required to return the PI3K signaling to a basal state.^(5, 6, 33) Inhibiting the PTEN and SHIP1 enzymatic regulation of PI3K allows for the control of the expansion of cell populations where these proteins are expressed, including but not limited to stem cell populations. However, PTEN's prominent role as a tumor suppressor, and particularly in prevention of myeloid leukemia development from the HSC compartment, appear to exclude PTEN inhibition as a drug target.^(3, 34) Isoform-specific PI3K agonists represent another potential avenue, however, as gain of function mutations in PI3K also contribute to malignancy, such investigations might be ill advised. In this regard, SHIP1 might be considered an optimal target for modulating the inositol phospholipid signaling therapeutically as it plays a prominent role in regulation of both the MSC and HSC compartments.^(5, 6, 8, 35) Moreover, targeting of SHIP1 in vivo does not result in the mucosal inflammatory disease observed in germline or induced SHIP1 mutant mice and the enzyme does not appear to have a role in suppressing hematologic malignancies including myeloid leukemia.^(13, 31, 36)

The soluble HSC niche components, such as G-CSF, can trigger proliferation, mobilization, and/or differentiation by the HSC compartment.^(25, 37-40) G-CSF binding to its receptor triggers activation of a PI3K mediated signaling cascade, promoting activation of both the MAP/ERK and AKT distal signaling arms. Our G-CSF neutralization studies indicate that the significant increase in BM cellularity, HSC numbers and MSC numbers promoted by SHIPi are primarily due to its induction of G-CSF. However, the impact on the HSC compartment, and perhaps also MSC, may also require attenuation of SHIP1 function at the G-CSFR in one or both stem cell populations. Thus, SHIPi may also increase PI3K signaling downstream of the G-CSFR in these cells and thereby further augment proliferation and survival of these primitive stem cells driven by the increased availability of soluble G-CSF in SHIPi treated hosts. This potential stem cell-intrinsic effect of SHIPi at the G-CSFR is likely critical for amplification of HSC by SHIPi, as administration of recombinant G-CSF alone is not sufficient to increase BM cellularity, HSC numbers and MSC frequency.^(21, 41)

An increase in PI3K/Akt signaling has a significant impact on transcription factors that up regulate genes important for survival and proliferation. An important example, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-□B), has been thoroughly studied in a PI3K/akt/SHIP context and has been shown to be a strong regulator of G-CSF activity.⁴²⁻⁴⁴ Activation of Akt by PI3K increases NF-κB signaling, while SHIP inhibits NF-κB activation in multiple cell types.^(45, 46) It stands to reason that cells responsible for the production of G-CSF utilize this pathway to increase secretion of G-CSF. Interestingly, a prominent role for PI3K signaling can be found in the majority of cell types responsible for the production of G-CSF, including monocytes, macrophages, and osteoblasts.⁴⁷⁻⁵² These studies suggest that the important intracellular role of the PI3K/Akt/NF-κB cascade could be responsible for the induction of G-CSF observed in SHIPi treated animals, and the subsequent effects on both the HSC and niche populations.

The capacity of SHIPi to expand both HSC and MSC in vivo suggests it could have a significant impact in diverse therapeutic settings that include bone marrow failure syndromes, aplastic anemia, myelofibrosis and radiation or chemotherapy-induced BM damage. The transient dosing regimen of 3AC utilized here allows for a beneficial increase in key soluble factors and cell populations that aid in BM recovery without triggering the life-threatening mucosal inflammation associated with long-term systemic ablation of SHIP1 in adults.¹³ Thus, it is feasible that periodic SHIPi treatment could be used to amplify the HSC and MSC compartments in diseases where the function of these stem cell populations are limited or in decline. This may have application beyond hematologic failure as MSC are also thought to play a role in promoting cardiovascular repair.^(53, 54) As SHIP1, or its stem cell analog, s-SHIP, may be expressed in other adult stem cell populations, utilization of SHIPi might be investigated in disease setting that would benefit from expansion of other adult tissue stem cells.^(55, 56)

Example 2 Therapeutic Use of SHIPi to Improve Expansion of HSCs and MSCs

Expansion or transplant of HSCs or MSCs can be useful in treating many diseases, including GVHD (graft versus host disease), suppression of graft rejection, suppression of autoimmunity, repair of myocardial infarcts, cardiovascular repair, drug-resistant polymyositis or dermatomyositis, multiple sclerosis (MS), amyotrophic lateral sclerosis, bone marrow failure syndromes (genetic or therapy induced) (HSC), cardiovascular diseases (MSC), heart repair (MSC), and possibly other diseases where MSC may mediate tissue repair or suppress immune responses.

For SHIPi in allogeneic or haploidentical HSCT: HSCs are removed from the donor (BM harvest, leukopheresis of mobilized blood). The patient is conditioned with a SHIP inhibitor daily (3AC, K118), delivered orally, via injection or another method used for administration of a therapeutic compound to an individual, for 6-7 days (could be done for fewer days (1, 2, 3, 4, 5) or more days (8, 10, 14, 21 etc.)). The patient may require additional mycloalbaltion (5FU, [fluorouacil], TBI [total body irradiation], cyclophosphamide) just prior to transplant to reduce competition from the host HSCs. Patient receives allo or haploidentical HSC transplant.

Example 3 Therapeutic Use of SHIPi to Induce Endogenous Production of Growth Factor G-CSF

Neutrophils play a critical role in combating bacterial and parasitic infections. Their numbers are increased by endogenous production of a growth factor G-CSF. Production of neutrophils from hematopoietic stem and progenitor cells is promoted by G-CSF.¹ This production is compromised by a wide variety of cancer and other therapies that damage the ability of stem cell and progenitors to produce neutrophils. Therefore recombinant G-CSF (Neupogen, Amgen or Teva Pharmaceuticals) is used clinically to boot neutrophil production to protect cancer patients who experience chemotherapy- or radiation-induced neutropenia and thus increased risk of life-threatening, opportunistic bacterial, fungal or parasitic infections.² G-CSF can also be used to boost immune protection in cases of severe infection of otherwise healthy individuals³ or in organ-transplant patients on immunosuppressive drugs.⁴ G-CSF is also quite effective at mobilizing of hematopoietic stem and progenitors cells from the bone marrow to the peripheral blood which increases the ease and yield of stem cell collection required for bone marrow transplantation, in either an autologous or allogeneic setting.¹ We have found that treatment of mice with SHIP1 inhibitors (SHIPi), either 3AC or K118, super-induces endogenous production of G-CSF, thus providing a small molecule drug type approach to induce the patient or donors own body to produce more G-CSF, obviating the need for administration of a highly costly recombinant protein that can be difficult to store and administer.

One aspect of the invention is the administration of a SHIP1-inhibiting compound to a patient suffering from a disease or condition for which the induction of G-CSF would have a therapeutic effect, including, but not limited to, patients suffering from diseases in which neutrophils play a role in protecting or curing the patient, such as in bacterial, fungal and parasitic infections, cancers, and chemotherapy- or radiation-induced neutropenia, organ transplant patients on immunosuppressive drugs, otherwise healthy patients with severe infections, and patients who will contribute tissue for bone marrow transplantation, for autografts or allografts. One aspect of the invention is the administration of a SHIP1 inhibiting compound to a patient who will benefit from the mobilization of hematopoietic or mesenchymal stem or progenitor cells into the peripheral blood, such as those with declining stem cell function associated with genetic, environmental or age-related factors. One aspect of the invention is the administration of a SHIP1 inhibiting compound to a patient to expand the patient's hematopoietic and/or mesenchymal stem cell compartments. Another aspect of the invention is the administration of a SHIP1 inhibiting compound to a patient suffering from a disease or condition for which the promotion of or accelerated recovery of white blood cell (WBC) and lymphocyte numbers will have therapeutic benefit, such as to patients after radiation-induced myeloablation.

K-118, 3AC and any of the other SHIP1-inhibiting compounds can be administered to such patients in the manner and dosing described elsewhere in the application. Water-soluble compounds can be administered orally or via injection, among other methods. Non-soluble therapeutic compounds.

REFERENCES

Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. All references cited herein are hereby incorporated by reference in their entirety. Certain references are cited by author and date, while other references are denoted by superscripted numerals. Below is a listing of various references cited herein, with the references being identified by author, date, publication, and page numbers:

-   1. Perry J M, He X C, Sugimura R, et al. Cooperation between both     Wnt/{beta}-catenin and PTEN/PI3K/Akt signaling promotes primitive     hematopoietic stem cell self-renewal and expansion. Genes Dev.     25:1928-1942. -   2. Polak R, Buitenhuis M. The PI3K/PKB signaling module as key     regulator of hematopoiesis: implications for therapeutic strategies     in leukemia. Blood. 119:911-923. -   3. Zhang J, Grindley J C, Yin T, et al. PTEN maintains     haematopoietic stem cells and acts in lineage choice and leukaemia     prevention. Nature. 2006;441:518-522. -   4. Kerr W G, A role for SHIP in stem cell biology and     transplantation. Curr Stem Cell Res Ther. 2008;3:99-106. -   5. Desponts, C, Hazen A L, Paraiso K H, et al. SHIP deficiency     enhances HSC proliferation and survival but compromises homing and     repopulation. Blood. 2006;107:4338-4345. -   6. Hazen A L, Smith M J, Desponts C, et al. SHIP is required for a     functional hematopoietic stem cell niche. Blood 2009;113:2924-2933. -   7. Helgason C D, Antonchuk J, Bodner C, et al. Homeostatis and     regeneration of the hematopoietic stem cell pool is altered in     SHIP-deficient mice. Blood. 2003. -   8. Iyer S, Viernes D R, Chisholm J D, et al. SHIP1 Regulates MSC     Numbers and Their Osteolineage Commitment by Limiting Induction of     the PI3K/Akt/beta-Catenin/Id2 Axis. Stem Cells Dev.     2014;23:2336-2351. -   9. Iyer S, Margulies B S, Kerr W G. Role of SHIP1 in bone biology.     Ann N Y Acad Sci. 2013;1280:11-14. -   10. Helgason C D, Damen J E, Rosten P, et al. Targeted disruption of     SHIP leads to hemopoietic perturbations, lung pathology, and a     shortened life span. Genes & Development. 1998;12:1610-1620. -   11. Ghansah T, Paraiso K H, Highfill S, et al. Expansion of myeloid     suppressor cells in SHIP-deficient mice represses allogeneic T cell     responses. J Immunol. 2004;173:7324-7330. -   12. Kerr W G, Park M Y, Maubert M, et al. SHIP deficiency causes     Crohn's disease-liek ileitis. Gut. 2011;60:177-188. -   13. Brooks, R Fuhler G M, Iyer S, et al. SHIP1 inhibition increases     immunoregulatory capacity and triggers apoptosis of hematopoietic     cancer cells. J Immunol. 2010;184:3582-3589. -   14. Fuhler G M, Brooks R, Toms B, et al. Therapeutic potential of     SH2 domain-containing inositol-5;-phosphatase 1 (SHIP1) and SHIP2     inhibition in cancer. Mol. Med. 2012;18:65-75. -   15. Harrison D E, Jordan C T, Zhong R K, et al. Primitive     hemopoietic stem cells: direct assay of most productive populations     by competitive repopulation with simple binomial, correlation and     covariance calculations. Experimental Hematology. 1993;21:206-219. -   16. Shojaei F, Wu X, Zhong C, et al. Bv8 regulates     myeloid-cell-dependent tumour angiogenesis. Nature 2007;450:825-831. -   17. Osawa M, Hanada K, Hamada H, et al. Long-term     lymphohematopoietic reconstitution by a single CD34-low/negative     hematopoietic stem cell. Science. 1996;273:242-245. -   18. Jordan C T, Astle C M, Zawadzki J, et al. Long-term repopulating     abilities of enriched fetal liver stem cells measured by competitive     repopulation. Experimental Hematology, 1995;23:1011-1015. -   19. Beerman I, Bhattacharya D, Zandi S, et al. Functionally distinct     hematopoietic stem cells modulate hematopoietic lineage potential     during aging by a mechanism of clonal expansion. Proc Natl Acad Sci     USA 107:5465-5470. -   20. Muller-Sicburg C E, Cho R H, Karlsson L, et al. Myeloid-biased     hematopoietic stem cells have extensive self-renewal capacity but     generate diminished lymphoid progeny with impaired IL-7     responsiveness. Blood 2004;103:4111-4118. -   21. Mendez-Ferrer S, Michurina T V, Ferraro F, et al. Mesenchymal     and haematopoietic stem cells form a unique bone marrow niche.     Nature 466:829-834. -   22. Zhou B O, Yue R, Murphy M M, et al. Leptin-receptor-expressing     mesenchymal stromal cells represent the main source of bone formed     by adult bone marrow. Cell Stem Cell. 15:154-168. -   23. Pinho S, Lacombe J, Hanoun M, et al. PDGFRalpha and CD51 mark     human nestin+ sphere-forming mesenchymal stem cells capable of     hematopoietic progenitor cell expansion. J Exp Med. 210:1351-1367. -   24. Morikawa S, Mabuchi Y, Kubota Y, et al. Prospective     identification, isolation, and systemic transplantation of     multipotent mesenchymal stem cells in murine bone marrow. J Exp Med.     2009;2016-2483-2496. -   25. Wilson A, Laurenti E, Oser G, et al. Hematopoietic stem cells     reversibly switch from dormancy to self-renewal during homeostasis     and repair. Cell. 2008;135:1118-1129. -   26. Morrison S J, Wright D H, Weissman I L.     Cyclophosphamide/granulocyte colony-stimulating factor induces     hematopoietic stem cells to proliferate prior to mobilization. Proc     Natl Acad Sci USA. 1997;94:1908-1913. -   27. Hunter M G, Avalos B R. Phosphatidylinositol 3′-kinase and     SH2-containing inositol phosphatase (SHIP) are recruited by distinct     positive and negative growth-regulatory domains in the granulocyte     colony-stimulating factor receptor. Journal of Immunology.     1998;160:4979-4987. -   28. Hunter M G, Jacob A, O'Donnell L C, et al. Loss of SHIP and CIS     recruitment ot the granulocyte colony-stimulating factor receptor     contribute to hyperproliferative responses in severe congenital     neutropenia/acute myelogenous leukemia. J. Immunol.     2004;173:5036-5045. -   29. Wang Y, Schulte B A, LaRue A C, et al. Total body irraidation     selectively induces murine hematopoietic stem cell senescence.     Blood. 2006;107:358-366. -   30. Viernes D R, Choi L B, Kerr W G, et al. Discovery and     development of small molecule SHIP phosphatase modulators. Med Res     Rev. 34:795-824. -   31. Kerr W G. Inhibitor and activator: dual functions for SHIP in     immunity and cancer. Ann N Y Acad Sci. 2011; 1217:1-17. -   32. Geest C R, Coffer P I. MAPK signaling pathways in the regulation     of hematopoiesis. J Leukoc Biol. 2009;86:237-250. -   33. Tesio M. Oser G M, Baccelli I, et al. Pten loss inthe bone     marrow leads to G-CSF-mediated HSC mobilization. J Exp Med.     210:2337-2349. -   34. Yilmaz O H, Valdez R, Theisen B K, et al. Pten dependence     distinguishes haematopoietic stem cells from leukaemia-initiating     cells. Nature. 2006;441:475-482. -   35. Barrett D M, Singh N, Porter D L, et al. Chimeric antigen     receptor therapy for cancer. Annual review of medicine.     2014;65:333-347. -   36. Maxwell M J, Srivastava N, Park M Y, et al. SHIP-1 deficiency in     the myeloid compartment is insufficient to induce myeloid expansion     or chronic inflammation. Genes Immun. 2014. -   37. Gibbs K D, Jr., Gilbert P M, Sachs K, et al. Single-cell     phospho-specific flow cytometric analysis demonstrates biochemical     and functional heterogeneity in human hematopoietic stem and     progenitor compartments. Blood. 117:4226-4233. -   38. Link D C. Mechanisms of granulocyte colony-stimulating     factor-induced hematopoietic progenitor-cell mobilization. Semin     Hematol. 2000;37:25-32. -   39. Hogge D E, Lansdorp P M, Reid D, et al. Enhanced detection,     maintenance, and differentiation of primitive human hematopoietic     cells in cultures containing murine fibroblasts engineered to     produce human steel factor, interleukin-3, and granulocyte     colony-stimulating factor. Blood. 1996;88:3765-3773. -   40. Bai L, Rohrschneider L R. s-SHIP promoter expression marks     activated stem cells in developing mouse mammary tissue. Genes Dev.     2010;24:1882-1892. -   41. Molineux G, Pojda Z, Dexter T M. A comparison of hematopoiesis     in normal and splenectomized mice treated with granulocyte     colony-stimulating factor. Blood 1990;75:563-569. -   42. O'Neill L A, Kaltschmidt C. NF-kappa B: a crucial transcription     factor for glial and neuronal cell function. Trends Neurosci.     1997;20:252-258. -   43. Baldwin A S. The NF-CE∫B AND ICE∫B PROTEINS: New Discoveries and     Insights. Annual Review of Immunology. 1996;14:649-681. -   44. Bar-Yehuda S, Madi L, Barak D, et al. Agonists to the A3     adenosine receptor induce G-CSF production via NF-CE∫B activation.     Experimental Hematology, 30:1390-1398. -   45. Conde C, Rambout X, Leburn M, et al. The inositol phosphatase     SHIP-1 inhibits NOD2-induced NF-kappaB activation by disturbing the     interaction of XIAP with RIP2. PLoS one. 7:341005. -   46. Li W, Wang H, Kuang C Y, et al. An essential role for the     Id1/PI3K/Akt/NFKB/survivin signalling pathway in promoting the     proliferation of endothelial progenitor cells in vitro. Mol Cell     Biochem. 363:135-145. -   47. Fujita T, Azuma Y, Fukuyama R, et a. Runx2 induces osteoblast     and chondrocyte differentian and enhances their migration by     coupling with PI3K-Akt signaling. J Cell Biol. 2004;166:85-95. -   48. Lieschke G J, Burgess A W. Granulocyte colony-stimulating factor     and granulocyte-macrophage colony-stimulating factor (2). N Engl J     Med. 1992;327:99-106. -   49. Taichman R S, Emerson S G. Human osteoblasts support     hematopiesis through the production of granulocyte     colony-stimulating factor. J Exp Med. 1994;179:1677-1682. -   50. Taichman R S, Reilly M J, Emerson S G. Human osteoblasts support     human hematopoietic progenitor cells in vitro bone marrow cultures.     Blood. 1996;87:518-524. -   51. Nioche S, Tazi A, Leeossier D, et al. Production of granulocyte     colony-stimulating factor (G-CSF) by human cells: T     lymphocyte-dependent and T lymphocyte-independent release of G-CSF     by blood monocytes. Eur J Immunol. 1988;18:1021-1026. -   52. Luyendyk J P, Schabbauer G A, Tencati M, et al. Genetic analysis     of the role of the PI3K-Akt pathway in lipopolysaccharide-induced     cytokine and tissue factor gene expression in monocytes/macrophages.     J Immunol. 2008;180:4218-4226. -   53. Fukuda K. Development of regenerative cariomyocytes from     mesenchymal stem cells for cardiovascular tissue engineering. Artif     Organs. 2001;25:187-193. -   54. Toma C, Pittenger M F, Cahill K S, et al. Human mesenchymal stem     cells differentiate to a cariomyocyte phenotype in the adult murine     heart. Circulation. 2002;105:93-98. -   55. Tu Z, Ninos J M, Ma Z, et al. Embryonic and hematopoietic stem     cells express a novel SH2-containing inositol 5′-phosphatase isoform     that partners with the Grb2 adapter protein. Blood.     2001;98:2028-2038. -   56. Bai L, Rohrschneider L R, s-SHIP promoter expression marks     activated stem cells in developing mouse mammary tissue. Genes Dev.     24:1882-1892.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. 

1. A method of inducing expression of granulocyte colony stimulating factor (G-CSF) in a subject, said method comprising: administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor to the subject.
 2. The method according to claim 1, wherein the SHIP1 inhibitor is a SHIP inhibitor compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein formula (I) is as follows:

wherein

at the 4,5 and 5,6 positions represents a single or double bond, with the proviso that the sum of double bonds present at the 4,5 and 5,6 positions is 0 or
 1. R¹ is a straight chain C1-C4 alkyl or C1-C4 haloalkyl; R² is hydrogen, methyl, or halomethyl; R³ and R¹³ (when present), are individually selected from hydrogen, substituted or unsubstituted amino, C1-C4 alkyl, C1-C4 haloalkyl, and alkenyl; R⁴ is hydrogen, hydroxy, substituted or unsubstituted amino, C1-C4 alkyl, or benzyl; R5 represents hydrogen or an alkyl group; X¹ is selected from the group consisting of hydrogen, hydroxy, mercapto, alkoxy, aryloxy, alkylthio, arylthio, alkylcarbonamido, alkoxycarbonamido, arylcarbonamido, aryloxycarbonamido, alkylsulfonamido, arylsulfonamido, substituted or unsubstituted amino, and aminoalkyl; and each X² individually represents a divalent oxo atom or two hydrogen atoms; with the proviso that X¹ cannot be a primary amino group when: R¹ and R² are each methyl; X², R³, R⁴, and R¹³ are each hydrogen; and R represents a 1, 5-dimethylhexyl alkyl group.
 3. The method according to claim 2, wherein at least one of X¹, R³, and R⁴ is substituted or unsubstituted amino.
 4. The method according to claim 3, wherein the substituted or unsubstituted amino is NH₂ or NH₃Cl.
 5. The method according to claim 2, wherein X¹ is NH₂ or NH₃Cl.
 6. The method according to claim 2, wherein said compound of formula (I) is a compound of a formula selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein X═NR₂, NRCOR, NHCONR₂, OR, SR, OCOR, OCONR₂, or NHCNHNH2, and wherein R═H, alkyl, cycloalkyl. aryl, or benzyl.
 7. The method according to claim 1, wherein the pan-SHIP1/2 inhibitor is a compound selected from the group consisting of:


8. The method according to claim 6, wherein the compound of Formula (I) or pharmaceutically acceptable salt thereof is a compound of Formula (IA) or a pharmaceutically acceptable salt thereof:

wherein

represents a single or double bond; R¹ and R² are individually selected from hydrogen and C₁-₃ alkyl; R³ selected from hydrogen and amino; R⁴ is selected from hydrogen, amino, and hydroxy; R⁵ is selected from hydrogen and C₁-₁₀ alkyl; and X¹ is selected from hydrogen, amino, and hydroxy.
 9. The method according to claim 8, comprising administering a hydrochloride salt of a compound of Formula (IA).
 10. The method according to claim 8, comprising administering a compound of one of the following Formulas (IB)-(IO), or a pharmaceutically acceptable salt thereof:


11. The method according to claim 1, wherein the SHIP1 inhibitor or the pan-SHIP1/2 inhibitor is a compound having a formula selected from the group consisting of:


12. The method according to claim 1, wherein the SHIP1 or pan-SHIP1/2 inhibitor is administered orally, rectally, intraperitoneally, transdermally, intradermally, topically, by inhalation, nasally, buccally, vaginally, via an implanted reservoir or by parenteral routes.
 13. The method according to claim 12, wherein the SHIP1 or pan-SHIP1/2inhibitor is injected intraperitoneally at between about 10 mg/kg and 80 mg/kg of body weight.
 14. The method according to claim 1, wherein administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor results in an increase in number of hematopoietic stem cells and/or mesenchymal stem cells in the subject.
 15. The method according to claim 1, wherein administering a safe and effective amount of a SHIP1 inhibitor or a pan-SHIP1/2 inhibitor results in an increase in number of neutrophils in the patient.
 16. The method according to claim 1, wherein the subject is suffering from a bacterial, fungal, or parasitic infection, a cancer, chemotherapy- or radiation-induced neutropenia, radiation-induced myeloablation, the effects of immunosuppressive drugs, or declining stem cell function associated with genetic, environmental or age-related factors.
 17. The method according to claim 1, wherein the SHIP1 inhibitor is either a small interfering RNA (siRNA) or a microRNA (miRNA) effective to inhibit SHIP1 via RNA interference (RNAi) (post transcriptional gene silencing). 